Alternative splicing regulation of gene expression and therapeutic methods

Abstract

To provide a medicine for treating a disease in a mammal.SOLUTION: Provided is a medicine for treating a disease in a mammal, comprising an expression cassette that is a first expression cassette comprising a chimeric gene operably linked to a first expression control element, the chimeric gene comprising a first portion comprising a minigene having an alternatively spliced exon and a second portion encoding a first RNA encoding a protein, the first portion being 5' to the second portion, the second portion lacking an initiation codon or start codon, the alternatively spliced exon comprising a translation initiation regulatory sequence, expression of the protein being controlled by inclusion of the alternatively spliced exon, and the medicine being used in combination with a small molecule splicing modifier that modulates splicing of the alternatively spliced exon.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 872,417, filed July 10, 2019, U.S. Provisional Application No. 62 / 838,223, filed April 24, 2019, U.S. Provisional Application No. 62 / 837,701, filed April 23, 2019, and U.S. Provisional Application No. 62 / 715,756, filed August 7, 2018, the entire contents of each of which are incorporated herein by reference.

[0002] 1. Field The present disclosure generally relates to the field of molecular biology and medicine. More specifically, the present disclosure relates to a method of using alternative splicing regulation to regulate the expression of target genes, such as those encoding inhibitory RNAs, therapeutic proteins, or parts of CRISPR / Cas9 systems. [Background technology]

[0003] 2. Description of Related Art Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder that primarily affects adults and occasionally children. HD is part of the polyglutamine (polyQ) disorder family, which includes at least nine different neurodegenerative disorders caused by the expansion of triplet CAG repeats in specific genes. 1,2 Although the HTT protein is ubiquitously expressed, the most affected tissue is the brain, with the striatum and motor cortex being the first to be affected. HD patients experience progressive neurodegeneration leading to death 10-20 years after disease onset. 2 There is no cure for HD, and current treatments are symptomatic. 3 Exciting early studies in HD animal models have demonstrated that reducing expression of mutant HTT ameliorates the disease, even when initiated after disease onset. 4,5 .

[0004] Several methods are used to lower HTT levels.6~10 , RNA interference (RNAi) is one of them. 8,9,11~14 RNAi is a biological process in which small RNA molecules regulate the expression of specific genes by translation inhibition or mRNA degradation. 15,16 Scientists have devised various methods to deliver RNAi triggers into cells, including small interfering RNA (siRNA), short hairpin RNA (shRNA) or artificial miRNA (amiRNA) (Figure 1). All of them have been used to efficiently reduce the expression of target genes after engaging the RNAi pathway. Reports have shown that reducing the levels of toxic polyQ proteins with RNAi ameliorates disease phenotypes in several polyglutamine repeat disease models. 8 , 11,12 These studies provide evidence that RNAi-based treatment of neurodegenerative diseases, including HD, is feasible. Short-term (6 weeks) data in non-human primates (NHPs) and other long-term (6 months) data in NHPs suggest that sustained RNAi therapy is safe. 17,18 .

[0005] However, repeated or lifelong administration of RNAi therapy to patients requires consideration of the possibility that unintended gene silencing and persistent co-opting of the cellular RNAi pathway may induce toxicity over time. Off-target silencing results from the interaction of the RNAi sequence with unintended, fully or partially complementary mRNA transcripts. 19~21 Although standard search algorithms can reduce the likelihood of perfect complementarity off-sequence silencing, it is difficult to avoid unintended silencing that occurs when there is partial complementarity between the expressed RNAi moiety and another sequence, resulting in miRNA-like repression. 19,22 In addition to off-target silencing, diversion of the RNAi pathway can also result in toxicity by saturating the cellular RNAi machinery and preventing endogenous miRNA regulation. 23This toxicity can be minimized if the trigger is delivered as an artificial miRNA sequence, which is processed more efficiently than shRNA. 24,25 These triggers enter the RNAi pathway before the initial Drosha / DGCR8 processing step. 15 Using weaker promoters (H1 or ApoE / hAAT promoters) can also reduce this type of toxicity. 26 Although these approaches have shown promise when tested in cells or in mice, it is difficult to predict whether sustained expression from these promoters over decades will be safe in humans. Therefore, an RNAi expression system that allows control over when and how much exogenous RNAi sequences are expressed in cells is far preferable to a constitutive expression platform. This regulated system is particularly important for human diseases where gene silencing is required throughout the life of the individual. Summary of the Invention

[0006] overview The present invention utilizes alternative splicing of pre-mRNA as a mechanism for regulating gene expression. Splicing of pre-mRNA is a post-transcriptional regulatory process that removes introns, resulting in protein diversity through the selective inclusion or exclusion of protein-coding exons, portions of exons, and alternative 5' and 3' non-coding exons. This alternative exon splicing can be used as a regulatory switch to control the production of specific proteins, such as mammalian transactivators designed to bind to upstream designer promoter sequences for non-coding RNA (e.g., siRNA) production or protein production. The present invention provides innovative methods for regulating the expression of non-coding RNAs and proteins in mammalian cells and mammalian subjects, such as humans, including humans with neurodegenerative diseases such as Huntington's disease and spinocerebellar ataxia, and humans with genetic defects, such as deficiencies in tripeptidyl peptidase 1 (TPP1).

[0007] Thus, the invention provides methods for controlling expression (i.e., modulating expression, such as increasing expression or decreasing expression) of non-coding RNAs and proteins in cells and subjects, e.g., mammalian cells and mammalian subjects.

[0008] In one aspect, the method comprises: A first expression cassette comprising a chimeric gene operably linked to a first expression control element, the chimeric gene comprising a first portion comprising a minigene that is subject to alternative splicing and a second portion encoding an RNA encoding a protein, wherein expression of the protein is controlled by alternative splicing of the first portion. to a cell, thereby providing and / or controlling expression of the protein.

[0009] In another embodiment, a method for providing a protein comprises the steps of: A first expression cassette comprising a chimeric gene operably linked to a first expression control element, the chimeric gene comprising a first portion comprising a minigene that is subject to alternative splicing and a second portion encoding an RNA encoding a protein, wherein expression of the protein is controlled by alternative splicing of the first portion. The method includes administering the method to a subject.

[0010] The present invention further provides methods of treating disease conditions such as neurodegenerative diseases, diseases caused by genetic defects, or diseases caused by defective gene expression.

[0011] In one embodiment, the method of treating a disease in a mammal comprises: A first expression cassette comprising a chimeric gene operably linked to a first expression control element, the chimeric gene comprising a first portion comprising a minigene that is subject to alternative splicing and a second portion encoding an RNA encoding a protein, wherein expression of the protein is controlled by alternative splicing of the first portion. to a mammal.

[0012] In some embodiments, the second portion, which encodes an RNA encoding a protein, contains a translation stop codon, lacks an initiation or start codon, is not an open reading frame for producing a protein, or encodes only a portion of a protein. In some embodiments, alternative splicing of the first portion alters the transcript to delete or disable a stop codon, introduce an initiation or start codon, restore an open reading frame, or provide a missing portion of a protein.

[0013] In some embodiments, the first portion is 5' to the second portion. In some embodiments, the first portion includes an in-frame translation stop codon. In some embodiments, alternative splicing of the first portion removes the translation stop codon.

[0014] In some embodiments, the protein is a transactivator protein. In some embodiments, the protein is not a reporter protein.

[0015] In one aspect, the method comprises: a first expression cassette comprising a chimeric gene operably linked to a first expression control element, the chimeric gene comprising a first portion comprising a minigene that is subject to alternative splicing and a second portion encoding a transactivator protein that binds to a second expression control element, wherein expression of the transactivator is controlled by alternative splicing of the first portion; and a second expression cassette comprising a nucleic acid sequence encoding an RNA operably linked to a second expression control element to which the transactivator protein binds; to the cell, thereby increasing expression of the RNA in the mammalian cell.

[0016] In another embodiment, the method for regulating expression of an RNA or protein comprises: a first expression cassette comprising a chimeric gene operably linked to a first expression control element, the chimeric gene comprising a first portion comprising a minigene that is subject to alternative splicing and a second portion encoding a transactivator protein that binds to a second expression control element, wherein expression of the transactivator is controlled by alternative splicing of the first portion; and a second expression cassette comprising a nucleic acid sequence encoding an RNA operably linked to a second expression control element to which the transactivator protein binds; to a subject, thereby increasing expression of the RNA in the subject.

[0017] In one embodiment, the method of treating a disease in a mammal comprises: a first expression cassette comprising a chimeric gene operably linked to a first expression control element, the chimeric gene comprising a first portion comprising a minigene that is subject to alternative splicing and a second portion encoding a transactivator protein that binds to a second expression control element, wherein expression of the transactivator is controlled by alternative splicing of the first portion; or a second expression cassette comprising a nucleic acid sequence encoding an RNA operably linked to a second expression control element to which the transactivator protein binds; to a mammal, thereby increasing expression of the RNA in the mammal and treating the disease.

[0018] In some embodiments, the RNA is an inhibitory RNA, such as an siRNA, shRNA, or miRNA. In some embodiments, the inhibitory RNA inhibits or reduces the expression of an aberrant or abnormal protein associated with a disease, thereby treating the disease.

[0019] In some embodiments, the RNA encodes a therapeutic protein. In some embodiments, the therapeutic protein corrects a protein defect associated with a disease, thereby treating the disease.

[0020] In some embodiments, the RNA encodes a Cas9 protein. In some embodiments, the method further comprises administering to the subject a third expression cassette comprising a nucleic acid sequence encoding a guide RNA operably linked to a third expression control element. In some embodiments, the third expression control element is a constitutive promoter. In some embodiments, expression of the Cas9 protein and the guide RNA corrects the genetic disease. In some embodiments, the Cas9 protein lacks nuclease function and expression of the Cas9 protein and the guide RNA inhibits expression of the gene.

[0021] In some embodiments, the first expression control element is a constitutive promoter, a cell type specific promoter, or an inducible promoter.

[0022] In some embodiments, the first portion of the first expression cassette and the second portion of the first expression cassette are separated by a cleavable peptide, hi some embodiments, the cleavable peptide is a self-cleaving peptide, a drug-sensitive protease, or a substrate for an endogenous endoprotease.

[0023] In some embodiments, the splicing of the alternatively spliced ​​minigene is regulated by a small molecule splicing modifier. In some embodiments, the splicing of the alternatively spliced ​​minigene is regulated by a disease state in a cell. In some embodiments, the splicing of the alternatively spliced ​​minigene is regulated by a cell type or tissue type.

[0024] In some embodiments, increased expression of the transactivator or protein is provided by inclusion of an alternatively spliced ​​exon in the first portion of the chimeric gene, in some embodiments, the included exon comprises a translation initiation regulatory sequence.

[0025] In some embodiments, increased expression of the transactivator or protein is provided by inclusion of SMN2 exon 7. In some embodiments, inclusion of SMN2 exon 7 is triggered by the presence of a small molecule splicing modifier. In some embodiments, the method further comprises administering a small molecule splicing modifier to the cell or subject, thereby increasing expression of the RNA or protein. In some embodiments, the small molecule splicing modifier is The file is TIFF2025060942000002.tif115128.

[0026] In some embodiments, increased expression of the transactivator or protein is provided by skipping of an alternatively spliced ​​exon in the first portion of the chimeric gene. In some embodiments, the skipped exon comprises a stop codon. In some embodiments, increased expression of the transactivator or protein is provided by skipping of MDM2 exons 4-11. In some embodiments, skipping of MDM2 exons 4-11 is triggered by the presence of a small molecule splicing modifier. In some embodiments, the small molecule splicing modifier is sudemycin.

[0027] In some embodiments, increased expression of the transactivator is conferred by the insertion of an exon into the transcript of the transactivator.

[0028] In some embodiments, increased expression of the transactivator is provided by exon skipping in the transcript of the transactivator.

[0029] In some embodiments, an exon is inserted into a transcript that encodes all or part of a protein, such as a therapeutic protein.

[0030] In some embodiments, the exon inserted into a transcript contains a sequence such that when introduced into the transcript, the exon restores a protein coding sequence or brings the protein coding sequence of the transcript in frame. Thus, introduction of the exon into a transcript allows a complete protein sequence to be encoded by the transcript, or the exon provides or restores an open reading frame in the transcript, thereby providing or restoring a sequence that is translated into a protein, such as a therapeutic protein.

[0031] In some embodiments, the exon inserted into the transcript contains a start or initiation codon (e.g., ATG) that is not present in the transcript. When the exon is introduced in frame into the transcript, this allows for translation of the encoded protein, e.g., a therapeutic protein.

[0032] In some embodiments, the exon inserted into the transcript deletes or neutralizes a translation stop codon in the transcript. When the exon is introduced into the transcript, the deletion or neutralization of the translation stop codon allows for translation of the encoded protein, e.g., a therapeutic protein.

[0033] In some embodiments, the first expression cassette or the second expression cassette is comprised in a viral vector.

[0034] In various embodiments, the disease is a neurodegenerative disease. In various embodiments, the neurodegenerative disease is a poly-glutamine repeat disease. In various embodiments, the poly-glutamine repeat disease comprises Huntington's disease (HD).

[0035] In various embodiments, the neurodegenerative disease is spinocerebellar ataxia (SCA).In certain aspects, SCA is any of SCA1, SCA2, SCA3, SCA4, SCA5, SCA6, SCA7, SCA8, SCA9, SCA10, SCA11, SCA12, SCA13, SCA14, SCA16, SCA17, SCA18, SCA19, SCA20, SCA21, SCA22, SCA23, SCA24, SCA25, SCA26, SCA27, SCA28 or SCA29.

[0036] In various embodiments, the administration is to the central nervous system (CNS). In certain aspects, the administration is to the brain. In certain aspects, the administration is to the ventricles.

[0037] In various embodiments, the first or second expression cassette or the expression cassette comprising the nucleic acid sequence encoding the protein comprises a viral vector. In certain aspects, the viral vector is selected from an adeno-associated viral (AAV) vector, a lentiviral vector, or a retroviral vector.

[0038] In various embodiments, the AAV vector comprises an AAV particle comprising AAV capsid proteins, and a first expression cassette or a second expression cassette or an expression cassette comprising a nucleic acid sequence encoding a protein is inserted between a pair of AAV inverted terminal repeats (ITRs). In certain aspects, the AAV capsid protein is derived from or selected from the group consisting of VP1, VP2 and / or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10 and AAV-2i8, or a capsid protein having 70% or greater identity to the VP1, VP2 and / or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-Rh10 or AAV-2i8. In certain aspects, one or more of the pair of ITRs is derived from, comprises, or consists of an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, or AAV-2i8, or an ITR having 70% or more identity to the ITR sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-Rh10, or AAV-2i8.

[0039] In various embodiments, the first expression cassette or the second expression cassette or the expression cassette comprising a nucleic acid sequence encoding a protein comprises a promoter.

[0040] In various embodiments, the first expression cassette or the second expression cassette or the expression cassette comprising a nucleic acid sequence encoding a protein comprises an enhancer element.

[0041] In various embodiments, the first expression cassette or the second expression cassette or the expression cassette comprising a nucleic acid sequence encoding a protein comprises a CMV enhancer or a chicken beta actin promoter.

[0042] In various embodiments, the first expression cassette or the second expression cassette or the expression cassette comprising a nucleic acid sequence encoding a protein further comprises one or more of an intron, a filler polynucleotide sequence and / or a polyA signal or a combination thereof.

[0043] In various embodiments, multiple AAV particles are administered.

[0044] In some embodiments, the AAV particles are about 1 × 10 6 ~Approx. 1×10 18 The drug is administered at a dose of 100 mg / kg.

[0045] In some embodiments, the AAV particles are about 1×10 7 ~1×10 17 , about 1×10 8 ~1×10 16 , about 1×10 9 ~1×10 15 , about 1×10 10 ~1×10 14 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 11, about 1×10 11 ~1×10 12 , about 1×10 12 ~×10 13 , or approximately 1 × 10 13 ~1×10 14 The vector is administered in a dose of genomes per kilogram of mammal (vg / kg).

[0046] In some embodiments, the AAV particles are 1×10 6 ~1×10 16 It is administered in a volume of approximately 0.5 to 4 ml of vg / ml.

[0047] In some embodiments, the method comprises administering a plurality of AAV empty capsids.

[0048] In some embodiments, the empty AAV capsid is formulated with the AAV particles administered to the mammal. In various aspects, the AAV empty capsid is administered or formulated with a 1.0-100-fold excess of AAV vector particles. In various aspects, the AAV empty capsid is administered or formulated with about a 1.0-100-fold excess of AAV empty capsid relative to the AAV particles.

[0049] In some embodiments, administration is by intraventricular and / or intraparenchymal injection.

[0050] In some embodiments, administration is to the ventricles, subarachnoid space and / or intrathecal space.

[0051] In some embodiments, administration is to nervous system cells, such as ependymal cells, pial cells, endothelial cells, ventricular cells, meningeal cells, glial cells, and / or neurons. In various aspects, the ependymal cells, pial cells, endothelial cells, ventricular cells, meningeal cells, glial cells, and / or neurons express RNAi.

[0052] In various embodiments, administration is to the rostral lateral ventricle, and / or the caudal lateral ventricle, and / or the right lateral ventricle, and / or the left lateral ventricle, and / or the right rostral lateral ventricle, and / or the left rostral lateral ventricle, and / or the right caudal lateral ventricle, and / or the left caudal lateral ventricle.

[0053] In various embodiments, administration is at a single site in the brain.

[0054] In various embodiments, administration is at 1 to 5 sites in the brain.

[0055] In various embodiments, administration is a single dose or multiple doses into either the cisterna magna, intraventricular space, ventricle, subarachnoid space, intrathecal space, and / or ependyma of a mammal.

[0056] In various embodiments, the method reduces adverse symptoms of Huntington's disease (HD) or spinocerebellar ataxia (SCA). In various aspects, the adverse symptoms are early or late symptoms, behavioral symptoms, personality or language symptoms, motor function symptoms, and / or cognitive symptoms.

[0057] In various embodiments, the methods increase, improve, maintain, restore or rescue memory deficits, memory impairment or cognitive function in a mammal.

[0058] In various embodiments, the methods ameliorate, inhibit, reduce, or prevent the worsening of loss of coordination, bradykinesia, or physical stiffness.

[0059] In various embodiments, the methods ameliorate, inhibit, reduce, or prevent the deterioration of spasm or fidgety movement.

[0060] In various embodiments, the methods ameliorate, inhibit, reduce, or prevent the worsening of depression or irritability.

[0061] In various embodiments, the methods ameliorate, inhibit, reduce, or prevent the worsening of dropping items, falls, loss of balance, difficulty speaking, or difficulty swallowing.

[0062] In various embodiments, the methods ameliorate, inhibit, reduce, or prevent the deterioration of the ability to organize.

[0063] In various embodiments, the methods ameliorate, inhibit, reduce, or prevent the worsening of ataxia or hyporeflexia.

[0064] In various embodiments, the methods ameliorate, inhibit, reduce, or prevent seizures or tremor seizures, or the worsening of tremor.

[0065] In various embodiments, the mammal is a non-rodent mammal. In various aspects, the non-rodent mammal is a primate. In various aspects, the primate is a human. In various aspects, the human is 50 years of age or older. In various aspects, the human is a child. In various aspects, the child is between about 1 year and about 8 years of age.

[0066] In various embodiments, the method includes administering one or more immunosuppressants. In various aspects, the immunosuppressant is administered before or simultaneously with the administration of the vector. In various aspects, the immunosuppressant is an anti-inflammatory agent.

[0067] As used herein, "essentially free" with respect to a specified component is used herein to mean that the specified component is not intentionally formulated in the composition and / or is present only as a contaminant or is present in only trace amounts. Therefore, the total amount of the specified component resulting from any unintentional contamination of the composition is much lower than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the specified component cannot be detected by standard analytical methods.

[0068] As used herein, "a" or "an" may mean one or more than one. As used in the claims, the words "a" or "an" when used in conjunction with the word "comprising" may mean one or more than one.

[0069] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer only to alternatives or the alternatives are mutually exclusive, however, the present disclosure supports both definitions that refer only to alternatives and definitions that refer to "and / or." As used herein, "another" can mean at least one more, or more.

[0070] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the instrument, the method being employed to determine the value, or the variation that exists among study subjects.

[0071] Other objects, features and advantages of the present invention will become apparent from the following detailed description, but it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are merely illustrative, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0072] In this specification, the present invention is broadly disclosed using affirmative language to describe numerous embodiments and aspects. The present invention also specifically includes embodiments in which certain content is completely or partially excluded, such as substances or materials, method steps and conditions, protocols, or procedures. For example, in certain embodiments or aspects of the present invention, materials and / or method steps are excluded. Thus, the present invention is generally not expressed in terms of what it does not include, but aspects that are not expressly excluded in the present invention are disclosed herein. [The present invention 1001] (a) a first expression cassette comprising a chimeric gene operably linked to a first expression control element, the chimeric gene comprising a first portion comprising a minigene that is subject to alternative splicing and a second portion encoding an RNA encoding a protein, expression of the protein being controlled by alternative splicing of the first portion; 23. A method for providing and / or regulating expression of a protein in a mammalian cell, comprising administering to the mammalian cell. [The present invention 1002] (a) a first expression cassette comprising a chimeric gene operably linked to a first expression control element, the chimeric gene comprising a first portion comprising a minigene that is subject to alternative splicing and a second portion encoding an RNA encoding a protein, expression of the protein being controlled by alternative splicing of the first portion; A method of providing a protein to a subject, comprising administering to the subject. [The present invention 1003] (a) a first expression cassette comprising a chimeric gene operably linked to a first expression control element, the chimeric gene comprising a first portion comprising a minigene that is subject to alternative splicing and a second portion encoding an RNA encoding a protein, expression of the protein being controlled by alternative splicing of the first portion; 23. A method of treating a disease in a mammal comprising administering to the mammal a [The present invention 1004] The method of claim 1003, wherein the second portion encoding the RNA encoding the protein contains a translation stop codon, lacks an initiation or start codon, is not an open reading frame for producing the protein, or encodes only a portion of the protein. [The present invention 1005] The method of claim 1004, wherein alternative splicing of the first portion modifies the transcript to delete or nullify a stop codon, introduce an initiation codon, restore an open reading frame, or provide a missing portion of a protein. [The present invention 1006] The method of any one of claims 1001 to 1005, wherein the first portion is located on the 5' side of the second portion. [The present invention 1007] The method of any of claims 1001 to 1006, wherein said first portion comprises an in-frame translation stop codon. [The present invention 1008] The method of claim 1007, wherein alternative splicing of said first portion removes a translation stop codon. [The present invention 1009] The method of any one of claims 1001 to 1008, wherein the protein is a transactivator protein. [The present invention 1010] the protein is a transactivator protein, and The method comprises the steps of: controlling expression of RNA in a mammalian cell; (b) a second expression cassette comprising a nucleic acid sequence encoding an RNA operably linked to a second expression control element to which the transactivator protein binds; to the cell, thereby increasing expression of the RNA in the mammalian cell. Further comprising: The method according to any one of 1001 and 1004 to 1009 of the present invention. [The present invention 1011] the protein is a transactivator protein, and The method comprises the steps of: regulating expression of RNA in a subject; (b) a second expression cassette comprising a nucleic acid sequence encoding an RNA operably linked to a second expression control element to which the transactivator protein binds; to a subject, thereby increasing expression of the RNA in the subject. Further comprising: The method of any one of 1002 and 1004 to 1009 of the present invention. [The present invention 1012] the protein is a transactivator protein, and The method comprises the steps of: treating a disease in a mammal, the method comprising: (b) a second expression cassette comprising a nucleic acid sequence encoding an RNA operably linked to a second expression control element to which the transactivator protein binds; to a mammal, thereby increasing expression of the RNA in the mammal and treating the disease. Further comprising: The method of any one of 1003 and 1004 to 1009 of the present invention. [The present invention 1013] The method of any one of claims 1010 to 1012, wherein the RNA is an inhibitory RNA. [The present invention 1014] The method of claim 1013, wherein said inhibitory RNA is an siRNA, shRNA, or miRNA. [The present invention 1015] The method of any one of claims 1013 to 1014, wherein said inhibitory RNA inhibits or reduces expression of an aberrant protein or proteins associated with a disease, thereby treating the disease. [The present invention 1016] The method of any of claims 1001 to 1015, wherein the RNA encodes a therapeutic protein. [The present invention 1017] The method of claim 1016, wherein said therapeutic protein corrects a protein defect associated with a disease, thereby treating the disease. [The present invention 1018] 16. The method of any one of claims 1001 to 1015, wherein the RNA encodes a Cas9 protein. [The present invention 1019] a third expression cassette comprising a nucleic acid sequence encoding a guide RNA operably linked to a third expression control element; The method of claim 1018, further comprising administering to a subject. [The present invention 1020] 1020. The method of claim 1019, wherein said third expression control element is a constitutive promoter. [The present invention 1021] The method of any one of claims 1019 to 1020, wherein expression of said Cas9 protein and said guide RNA corrects a genetic disease. [The present invention 1022] 1021. The method of any of claims 1018 to 1020, wherein the Cas9 protein lacks a nuclease function, and expression of the Cas9 protein and the guide RNA inhibits expression of a gene. [The present invention 1023] The method of any of claims 1001 to 1022, wherein said first expression control element is a constitutive promoter, a cell type specific promoter, or an inducible promoter. [The present invention 1024] The method according to any of claims 1001 to 1023, wherein the first part of said first expression cassette and the second part of said first expression cassette are separated by a cleavable peptide. [The present invention 1025] The method of claim 1024, wherein said cleavable peptide is a self-cleaving peptide, a drug-sensitive protease, or a substrate for an endogenous endoprotease. [The present invention 1026] The method of any of claims 1001 to 1025, wherein splicing of said alternatively spliced ​​minigene is regulated by a small molecule splicing modifier. [The present invention 1027] The method of any of claims 1001 to 1025, wherein splicing of said alternatively spliced ​​minigene is regulated by a disease state in a cell. [The present invention 1028] The method of any of claims 1001 to 1025, wherein splicing of said alternatively spliced ​​minigene is regulated by cell type or tissue type. [The present invention 1029] The method of any of claims 1001 to 1028, wherein the increased expression of said transactivator or said protein is provided by inclusion of an alternatively spliced ​​exon in the first part of the chimeric gene. [The present invention 1030] The method of claim 1029, wherein the included exon comprises a translation initiation regulatory sequence. [The present invention 1031] The method of any of claims 1001 to 1030, wherein the increased expression of said transactivator or said protein is provided by inclusion of SMN2 exon 7. [The present invention 1032] The method of claim 1031, wherein said SMN2 exon 7 inclusion is triggered by the presence of a small molecule splicing modifier. [The present invention 1033] administering said small molecule splicing modifying agent to said cell or said subject, thereby increasing expression of said RNA or said protein. The method of the present invention 1032 further comprises: [The present invention 1034] The small molecule splicing modifying agent is The method of the present invention 1032 or 1033, which is TIFF2025060942000003.tif122149. [The present invention 1035] The method according to any of claims 1001 to 1028, wherein increased expression of said transactivator or said protein is provided by skipping of an alternatively spliced ​​exon in the first part of the chimeric gene. [The present invention 1036] 1035. The method of claim 1035, wherein the skipped exon comprises a stop codon. [The present invention 1037] The method of any of claims 1001 to 1036, wherein the increased expression of said transactivator or said protein is provided by skipping of MDM2 exons 4 to 11. [The present invention 1038] The method of the present invention 1037, wherein skipping of MDM2 exons 4-11 is triggered by the presence of a small molecule splicing modifier. [The present invention 1039] The method of claim 1038, wherein the small molecule splicing modifying agent is sudemycin. [The present invention 1040] The method according to any one of claims 1001 to 1039, wherein said first expression cassette or said second expression cassette is contained in a viral vector. [The present invention 1041] 104. The method of claim 1040, wherein said viral vector is selected from an adeno-associated viral (AAV) vector, a lentiviral vector, or a retroviral vector. [The present invention 1042] The method according to any one of claims 1003 to 1006 and 1010 to 1041, wherein the disease is caused by a protein deficiency. [The present invention 1043] The method according to any one of claims 1003 to 1006 and 1010 to 1041, wherein the disease is caused by a genetic defect. [The present invention 1044] The method according to any one of claims 1003 to 1006 and 1010 to 1041, wherein the disease is a neurodegenerative disease. [The present invention 1045] The method of claim 1044, wherein the neurodegenerative disease comprises a poly-glutamine repeat disease. [The present invention 1046] The method of claim 1045, wherein the poly-glutamine repeat disease comprises Huntington's disease (HD). [The present invention 1047] The method of claim 1044, wherein said neurodegenerative disease comprises spinocerebellar ataxia (SCA), optionally any of SCA1 to SCA29. [The present invention 1048] The method according to any one of claims 1001 to 1047, wherein the mammal is a human. [The present invention 1049] The method according to any one of claims 1002 to 1006 and 1008 to 1048, wherein the administration is to the central nervous system. [The present invention 1050] The method according to any one of claims 1002 to 1006 and 1008 to 1049, wherein the administration is to the brain. [The present invention 1051] The method of claim 1050, wherein said administering is to a cerebral ventricle. [The present invention 1052] Any of the methods of claims 1041 to 1051, wherein the AAV vector comprises an AAV particle containing an AAV capsid protein, and the first expression cassette or the second expression cassette is inserted between a pair of AAV inverted terminal repeats (ITRs). [The present invention 1053] 1052. The method of claim 1052, wherein said AAV capsid protein is derived from or selected from the group consisting of VP1, VP2 and / or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10 and AAV-2i8, or capsid proteins having 70% or more identity to VP1, VP2 and / or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-Rh10 or AAV-2i8. [The present invention 1054] 1052. The method of claim 1052, wherein one or more of the pair of ITRs is derived from, comprises or consists of an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10 or AAV-2i8, or an ITR having 70% or more identity to an ITR sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-Rh10 or AAV-2i8. [The present invention 1055] The method of any one of claims 1001 to 1054, wherein said first expression cassette or said second expression cassette comprises a promoter. [The present invention 1056] The method of any one of claims 1001 to 1055, wherein said first expression cassette or said second expression cassette comprises an enhancer element. [The present invention 1057] The method of any one of claims 1001 to 1055, wherein said first expression cassette or said second expression cassette comprises a CMV enhancer or a chicken β-actin promoter. [The present invention 1058] Any of the methods of claims 1001 to 1057, wherein the first expression cassette or the second expression cassette further comprises one or more of an intron, a filler polynucleotide sequence and / or a polyA signal or a combination thereof. [The present invention 1059] The method of any of claims 1041 to 1058, wherein a plurality of viral vectors are administered. [The present invention 1060] The viral vector is about 1×10 6 ~Approx. 1×10 18 The method of the present invention, wherein the vector is administered in a dose of vector genome per kilogram (vg / kg). [The present invention 1061] The viral vector is about 1×10 7 ~1×10 17, about 1×10 8 ~1×10 16 , about 1×10 9 ~1×10 15 , about 1×10 10 ~1×10 14 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 11 , about 1×10 11 ~1×10 12 , about 1×10 12 ~×10 13 , or approximately 1 × 10 13 ~1×10 14 The method of the present invention, wherein the compound is administered in a dose of 1059 vg / kg mammal. [The present invention 1062] The viral vector is 1×10 6 ~1×10 16 The method of claim 1059, wherein the amount of 1000 vg / ml is administered in a dose of about 0.5 to 4 ml. [The present invention 1063] The method of any one of claims 1041 to 1062, further comprising the step of administering a plurality of empty viral capsids. [The present invention 1064] The method of claim 1063, wherein said empty viral capsids are formulated together with the viral particles that are administered to a mammal. [The present invention 1065] The method of claim 1063 or 1064, wherein said empty viral capsids are administered or formulated with a 1.0 to 100 fold excess of viral vector particles or empty viral capsids. [The present invention 1066] The method of claim 1063 or 1064, wherein said empty viral capsids are administered or formulated with about a 1.0-100 fold excess of empty viral capsids relative to viral vector particles. [The present invention 1067] The method of any of claims 1001 to 1066, wherein said delivering or said administering comprises intraventricular injection and / or intraparenchymal injection. [The present invention 1068] The method of any of claims 1001 to 1068, comprising administering or delivering it to the ventricles, the subarachnoid space, and / or the intrathecal space. [The present invention 1069] The method of any of claims 1001 to 1068, wherein said cells comprise ependymal cells, pial cells, endothelial cells, ventricular cells, meningeal cells, glial cells, and / or neurons. [The present invention 1070] The method of claim 1069, wherein said ependymal cells, said pial cells, said endothelial cells, said ventricular cells, said meningeal cells, said glial cells, and / or said neurons express said RNA or said protein. [The present invention 1071] The method according to any one of claims 1003 to 1006 and 1010 to 1070, wherein the administration is to a single site in the brain. [The present invention 1072] The method according to any one of claims 1003 to 1006 and 1010 to 1070, wherein the administration is at 1 to 5 sites in the brain. [The present invention 1073] Any of the methods of the present inventions 1003 to 1006 and 1010 to 1070, wherein the administration is to the rostral lateral ventricle, and / or the caudal lateral ventricle, and / or the right lateral ventricle, and / or the left lateral ventricle, and / or the right rostral lateral ventricle, and / or the left rostral lateral ventricle, and / or the right caudal lateral ventricle, and / or the left caudal lateral ventricle. [The present invention 1074] The method of any one of claims 1003 to 1006 and 1010 to 1070, wherein the administration is a single or multiple administration into any of the cisterna magna, intraventricular cavity, ventricle, subarachnoid space, intrathecal space, and / or ependyma of a mammal. [The present invention 1075] The method of any of claims 1003-1006 and 1010-1070, which reduces adverse symptoms of Huntington's disease (HD) or spinocerebellar ataxia (SCA). [The present invention 1076] The method of any one of claims 10 to 15, wherein the adverse symptoms include early or late symptoms, behavioral symptoms, personality or language symptoms, motor function symptoms, and / or cognitive symptoms. [The present invention 1077] The method of any of claims 1002 to 1006 and 1008 to 1076, which increases, improves, maintains, restores, or rescues memory deficit, memory impairment, or cognitive function in said mammal. [The present invention 1078] The method of any of inventions 1002-1006 and 1008-1077, which ameliorates, inhibits, reduces, or prevents the worsening of loss of coordination, slowness of movement, or physical stiffness. [The present invention 1079] The method of any of claims 1002-1006 and 1008-1078, which ameliorates, inhibits, reduces or prevents deterioration of spasms or fidgety movement. [The present invention 1080] The method according to any one of claims 1002 to 1006 and 1008 to 1079, which ameliorates, inhibits, reduces, or prevents the worsening of depression or irritability. [The present invention 1081] The method of any of inventions 1002-1006 and 1008-1080, which ameliorates, inhibits, reduces, or prevents the worsening of dropping items, falls, loss of balance, difficulty speaking, or difficulty swallowing. [The present invention 1082] The method of any of the present inventions 1002 to 1006 and 1008 to 1081, which improves, inhibits, reduces, or prevents the deterioration of the ability to organize. [The present invention 1083] The method of any one of claims 1002 to 1006 and 1008 to 1082, which ameliorates, inhibits, reduces, or prevents the worsening of ataxia or hyporeflexia. [The present invention 1084] Seizures or Tremors Any of the methods of inventions 1002-1006 and 1008-1083, which ameliorates, inhibits, reduces, or prevents the worsening of seizures or tremors. [The present invention 1085] The method according to any one of claims 1001 to 1084, wherein the mammal is a non-rodent mammal. [The present invention 1086] The method of claim 1085, wherein said non-rodent mammal is a primate. [The present invention 1087] The method of claim 1086, wherein the primate is a human. [The present invention 1088] The method of claim 1087, wherein the human is 50 years of age or older. [The present invention 1089] The method of claim 1088, wherein the human is a child. [The present invention 1090] The method of claim 1089, wherein the child is from about 1 year old to about 8 years old. [The present invention 1091] The method according to any one of claims 1003 to 1006 and 1010 to 1090, further comprising the step of administering one or more immunosuppressants. [The present invention 1092] The method of claim 1091, wherein said immunosuppressant is administered prior to or simultaneously with administration or delivery of the vector. [The present invention 1093] The method of claim 1091, wherein said immunosuppressant is an anti-inflammatory agent. [Brief description of the drawings]

[0073] The drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0074] [Figure 1] 1 depicts the repurposing of the RNAi pathway to silence expression in mammalian cells. [Diagram 2] Representing the action of a regulated promoter system. Inclusion of exon 7 of SMN2 is induced by LMI070, which allows translation of the e6 / 7 / 8 transactivator. [Diagram 3] The optimized promoter constructs respond to transactivators. Figure 3a shows that transactivator binding to RNAip induces luciferase expression. Figure 3b shows that the promoter variants tested (TA, TF2, TF4 and TF5) show negligible expression, comparable to control cells transfected with an empty promoter plasmid (NO), and are fully activated in response to transactivation, as determined by luciferase activity 24 hours after transfection of HEK293 cells. [Figure 4] Modified SMN2 / transactivator minigenes express spliced ​​RNA transcript isoforms that either constitutively exclude exon 7 (CSI3) or constitutively include exon 7 (CSI5), affecting background expression of optimized RNAi promoters. Figure 4a shows that transactivators were cloned downstream of a self-cleaving 2A peptide and SMN2 minigenes containing exons 6-7 and the 5' end of exon 8 and the minimal intronic intervening sequence required to recapitulate SMN2 splicing. The 3' and 5' exon 7 splice sites in the SMN2 / transactivator minigenes were modified to either constitutively exclude exon 7 (CSI3, 3' modified) or constitutively include exon 7 (CSI5, 5' modified). Figure 4b shows that for CSI, 10% of transcripts include exon 7, whereas for CSI3 and CSI5, exon 7 is either included or excluded. FIG. 4c shows that a modification that constitutively eliminates exon 7 (CSI3, a 3′ modification) minimized background activation of the RNAi promoter. [Diagram 5]Splicing and activity of the CSI and CSI3 minigenes in response to LIM070. Figure 5a shows exon 7 inclusion determined by semi-quantitative RT-PCR after 20 hours of LMI070 treatment in HEK293 cells. Figure 5b shows activation of the TF5 RNAi promoter in response to LMI070 determined by luciferase activity after 20 hours of treatment in HEK293 cells co-transfected with the SMN2 minigene and the TF5 RNAi promoter plasmid. [Figure 6] 6A-6B show quantitative assessment of HTT silencing by mi2.4v1, miHDS1v6a and their seed match miRNA controls in HEK293 after transfection: Figure 6A shows Q-PCR (24 hours) and Figure 6B shows Western blot (48 hours). [Figure 7] Figure 1 depicts a model of LMI070-regulated RNAi. Administration of LMI070 will induce miRNA expression and mHTT knockdown (black, RNAi effect from a single dose of LM1070). RNAi expression should peak 24-48 hours after LMI070 dosing, after which the artificial miRNA will taper off to background levels. Expected RNAi expression over a one-week period is shown after a single (red) or double (blue dashed) dose of LMI070. [Figure 8] De novo splicing of HTT in response to LMI070. RNAseq data from HEK293 cells treated with DMSO (CTL) or LMI070 (25 nM). Sashimi plot depicts novel minigenes identified by RNA-seq (inside the circle in the treated samples). [Figure 9] De novo splicing of beclin 1 (BECN1) in response to LMI070. RNAseq data from HEK293 cells treated with DMSO (CTL) or LMI070 (25 nM). Sashimi plot depicts novel minigenes identified by RNA-seq (inside the circle in the treated samples). [Figure 10] De novo splicing of chromosome 12 open reading frame 4 (C12orf4) in response to LMI070. RNAseq data from HEK293 cells treated with DMSO (CTL) or LMI070 (25 nM). Sashimi plot depicts novel minigenes identified by RNA-seq (inside the circle in the treated samples). [Figure 11] De novo splicing of 5'-3' exoribonuclease 2 (XRN2) in response to LMI070. RNAseq data from HEK293 cells treated with DMSO (CTL) or LMI070 (25 nM). Sashimi plot depicts novel minigenes identified by RNA-seq (inside the circle in the treated samples). [Figure 12] De novo splicing of splicing factor 3b subunit 3 (SF3B3) in response to LMI070. RNAseq data from HEK293 cells treated with DMSO (CTL) or LMI070 (25 nM). Sashimi plot depicts novel minigenes identified by RNA-seq (inside the circle in the treated samples). [Figure 13] De novo splicing of formin homology 2 domain containing 3 (FHOD3) in response to LMI070. RNAseq data from HEK293 cells treated with DMSO (CTL) or LMI070 (25 nM). Sashimi plot depicts novel minigenes (inside the circle in treated samples) identified by RNA-seq. [Figure 14] De novo splicing of glucosyltransferase 1 (GXYLT1) in response to LMI070. RNAseq data from HEK293 cells treated with DMSO (CTL) or LMI070 (25 nM). Sashimi plot depicts novel minigenes (inside the circle in treated samples) identified by RNA-seq. [Figure 15]De novo splicing of pyridoxal dependent decarboxylase domain containing 1 (PDXDC1) and nuclear pore complex interacting protein (PDXDC2P-NPIPB14P) non-coding RNAs in response to LMI070. RNAseq data from HEK293 cells treated with DMSO (CTL) or LMI070 (25 nM). Sashimi plot depicts novel minigenes identified by RNA-seq (inside the circle in treated samples). [Figure 16] De novo exon splicing induced by LMI070 (25 nM) in HEK293 cells. Inclusion of novel exons (identified by RNA-seq) in response to LMI070 treatment is confirmed by PCR. [Figure 17] Figure 1 depicts the XonSwitch strategy used to control gene expression. In the absence of a splice modifier, the new exon is excluded and the downstream protein is out-of-frame; therefore, in this case, no protein is made. After treatment with a splice modifier, the open reading frame is restored and the downstream protein is produced. [Figure 18] Represents XonSwitch strategy 2 used to control gene expression. The upstream ATG translation start codon was deleted and inserted within a novel pseudoexon. In the absence of the splice modifier, no translation occurs as the ATG translation start codon is not present in the transcript, and only non-protein-coding RNA is produced. Upon addition of the splice modifier, the pseudoexon includes the ATG start codon in the transcript, which is translated to express the protein. This provides tight regulation of protein expression. [Figure 19] Figure 1 depicts splicing of the SMN2 minigene in response to the splice modifier RG7800. PCR splicing assay depicting induction of exon 7 inclusion in the SMN2 minigene in response to splice modifier (RG7800) treatment at various doses (10 nM, 100 nM, 1 μM and 10 μM). [Figure 20] Figure 1 depicts inducible CRISPR epigenetic silencing by regulated Cas9 expression in response to splice modifier (RG7800) treatment. In the absence of RG7800, Cas9 protein is out-of-frame and not expressed. Treatment with RG7800 induces inclusion of exon 7 and restores Cas9 expression. Cas9 expression in response to RG7800 results in the formation of a specific Cas9 / sgRNA / RBP-Krab complex that binds to the HTT promoter, inducing epigenetic silencing of mutant HTT alleles. [Figure 21] The figure represents the regulatory editing of mHTT allele. Western blot represents HTT epigenetic silencing induced by SMN2_CRISPRi regulatory system in response to splice modifier (RG7800). Shown are HTT protein levels and Cas9 protein levels after treatment with RG7800 (1 μM) in HEK293 cells transfected with SMN2_CRISPRi system. As shown in the figure, Cas9 protein levels are increased in response to RG7800 and HTT expression levels are reduced by approximately 45% as a result of Cas9-mediated epigenetic silencing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] Detailed Description Disclosed herein is a chimeric transactivator minigene, in which alternative splicing of the minigene determines whether the downstream transactivator is expressed. The expression of the transactivator results in the transcription of the target gene under the control of the designer promoter sequence. The target gene can code for an inhibitory RNA, a CRISPR-Cas9 protein, or a therapeutic protein.

[0076] In one example, the minigene contains three exons, exons 1-3, with exon 2 skipped in the basal state. When exon 2 is skipped, the reading frame of exon 3 is shifted, resulting in the creation of a nonsense mutation in exon 3. Thus, translation of the encoded protein stops in exon 3, and nothing downstream is translated. Since the transactivator coding sequence is located downstream of the minigene, the transactivator is not expressed in the basal state. Alternatively, the translation initiation regulatory sequence is located in exon 2, and therefore no translation occurs when exon 2 is skipped.

[0077] To turn on expression of the transactivator, inclusion of the skipped exon must be induced. This can occur as a result of the presence of a small molecule splicing modifier. For example, a minigene can contain exons 6-8 of the SMN2 gene. In this case, exon 7 is skipped in the basal state. However, in the presence of certain splicing modifier small molecules (e.g., LMI070), exon 7 is included (see Figure 2). 31 Thus, the downstream transactivator will be expressed in the presence of LMI070 and not expressed in its absence.

[0078] Alternatively, inclusion of the skipped exon may be induced in one cell type but not in another. For example, exon 8 and exon 9 of FGFR2 are mutually exclusive, and only exon 9 is included in mesenchymal tissues (Takeuchi et al., 2010). Thus, the minigene may contain exons 7, 8, 9, and 10 of the FGFR2 gene to allow expression of the transactivator only in mesenchymal cells. In this case, a stop codon is engineered in exon 8 of the minigene to prevent transactivator expression in non-mesenchymal cells.

[0079] Further examples of cell type-specific alternative splicing events that can be used in the expression control system of the present disclosure include Eps8 exon 18B (chr12:15,792,360-15,792,395) and Eps8 exon 18C (chr12:15,787,673-15,787,696), which are specific to auditory hair cells.

[0080] Alternatively, inclusion of skipped exons can be induced by certain disease states, such as Huntington's disease, which results in the generation of transcript isoforms generated by alternative splicing. 32 Thus, the minigene could contain an exon from a gene whose splicing is altered in Huntington's disease, i.e., a gene in which an exon that is normally skipped in healthy cells is instead altered to be included when mutant HTT is expressed, such as PCDH1 (5:141869432-141878222). If necessary, a stop codon could be engineered into the alternatively spliced ​​exon to ensure that the transactivator is not produced in non-diseased cells. As a result, the transactivator will only be expressed if mutant HTT is present. In this example, the target The target gene may code for an inhibitory RNA that knocks down the expression of mutant HTT, thus creating an autoregulatory feedback loop. That is, the presence of mutant HTT reduces mutant HTT levels to a level that reverts splicing of the minigene to a non-diseased state by inducing expression of an inhibitory RNA that targets mutant HTT, thereby turning off expression of the inhibitory RNA and allowing expression of mutant HTT, which reaches a level that induces expression of the inhibitory RNA, and so on. Alternatively, the target gene may code for a CRISPR-Cas9 system that suppresses transcription of the HTT gene.

[0081] In another example, a minigene contains three exons, exons 1-3, and in the basal state, exon 2 is included. Inclusion of exon 2 can result in a downstream frameshift such that translation stops at exon 3, or exon 2 can be engineered to contain a stop codon. Thus, when exon 2 is included, i.e., in the basal state, the transactivator is not expressed.

[0082] To turn on the expression of the transactivator, the skipping of exon 2 must be induced. It can occur as a result of the presence of a small molecule splicing modifier. For example, the minigene can include exons 3, 4, 10, 11, and 12 of the MDM2 gene (Singh et al., 2009), all of which are included in the basal state. However, in the presence of certain splicing modifier small molecules (e.g., sudemycin), exons 4, 10, and 11 are skipped (Shi et al., 2015). Thus, the downstream transactivator will be expressed in the presence of sudemycin and not expressed in its absence. In this case, a stop codon may be engineered into exon 4 of the minigene to ensure that no protein is produced in the absence of sudemycin.

[0083] Alternatively, skipping of selectively included exons may be induced in some cell types but not in others. Exon 18 of Nin is skipped in neurons (Zhang et al., 2016). Thus, a minigene may contain exons 17, 18, and 19 of the Nin gene to allow expression of the transactivator only in neurons. In this case, a stop codon is engineered in exon 18 of the minigene to prevent transactivator expression in non-neuronal cells where exon 18 is included.

[0084] Alternatively, skipping of alternatively included exons can be induced by certain disease states, e.g., Huntington's disease, which results in the generation of transcript isoforms generated by alternative splicing. 32 Thus, the minigene may contain exons from genes whose splicing is altered in Huntington's disease, i.e., exons that are normally included in healthy cells are instead skipped when mutant HTT is expressed. To ensure that the transactivator is not produced in non-diseased cells, a stop codon may be engineered into an exon downstream of the alternatively spliced ​​exon. As a result, the transactivator will only be expressed if mutant HTT is present. In this example, the target gene may encode an inhibitory RNA that knocks down the expression of mutant HTT, thus creating an autoregulatory feedback loop. That is, the presence of mutant HTT reduces mutant HTT levels to a level that reverts splicing of the minigene to the non-diseased state, by inducing expression of an inhibitory RNA that targets mutant HTT, thereby turning off expression of the inhibitory RNA and allowing expression of mutant HTT, which reaches a level that induces expression of the inhibitory RNA, and so on. Alternatively, the target gene may encode a CRISPR-Cas9 system that suppresses transcription of the HTT gene.

[0085] Expression of the chimeric transactivator minigene can be regulated by various types of promoters depending on the desired expression pattern. For example, the promoter can be a universal constitutive promoter, such as the promoter of a housekeeping gene (e.g., ACTB). As another example, the promoter can be a cell type specific promoter, such as the promoter of synapsin in the case of neuronal expression. As yet another example, the promoter can be an inducible promoter.

[0086] The chimeric transactivator minigene may have a cleavable peptide located between the minigene and the transactivator. Optionally, the cleavable peptide may be a self-cleavable peptide, such as, for example, a 2A peptide. The 2A peptide may be a T2A peptide, a P2A peptide, an E2A peptide, or an F2A peptide. The presence of this peptide will result in the minigene-encoded peptide being separated from the transactivator protein after translation. Optionally, the cleavable peptide may be the cleavage site of a widely expressed endogenous endoprotease, such as furin, prohormone convertase 7 (PC7), paired basic amino-acid cleaving enzyme 4 (PACE4), or subtilisin kexin isozyme 2 (SKI-1). In some cases, the cleavable peptide may be a cleavage site for a tissue-specific or cell-specific endoprotease, such as prohormone convertase 2 (PC2; expressed primarily in endocrine tissues and the brain), prohormone convertase 1 / 3 (PC1 / 3; expressed primarily in endocrine tissues and the brain), prohormone convertase 4 (PC4; expressed primarily in the testis and ovary), and proprotein convertase subtilisin kexin 9 (PSCK9; expressed primarily in the lung and liver).

[0087] Also disclosed herein is a chimeric transactivator minigene, in which the minigene is inserted into the coding sequence of the transactivator, and alternative splicing of the minigene determines whether the transactivator is expressed. For example, an MDM2 minigene containing exons 4, 10, and 11 can be inserted into the coding sequence of the transactivator (Shi et al., 2015). In the basal state, the inclusion of the minigene exons disrupts the transactivator coding sequence. To ensure that no harmful protein is produced when the minigene exons are incorporated, the chimeric transactivator minigene can be engineered such that the minigene portion is located at or near the 5' end of the transactivator coding sequence, and a stop codon can be engineered into exon 4 of the minigene. To induce expression of the transactivator, a splicing modifier molecule (e.g., sudemycin) is used to induce skipping of exons 4, 10, and 11 of the MDM2 minigene. Similarly, cell type-specific or disease state alternative splicing events may be used as minigenes inserted into the coding sequence of the transactivator.

[0088] Also disclosed herein is a chimeric target gene minigene, in which the minigene is inserted into the coding sequence of the target gene, and alternative splicing of the minigene determines whether the target gene is expressed. The target gene may code for an inhibitory RNA, a CRISPR-Cas9 protein, or a therapeutic protein. For example, an MDM2 minigene containing exons 4, 10, and 11 may be inserted into the coding sequence of the target gene (Shi et al., 2015). In the basal state, the inclusion of the minigene exons disrupts the target gene coding sequence. To ensure that no harmful protein is produced when the minigene exons are incorporated, the chimeric transactivator minigene may be engineered such that the minigene portion is placed at or near the 5' end of the target gene coding sequence, and a stop codon may be engineered into exon 4 of the minigene. To induce expression of the target gene, a splicing modifier molecule (e.g., sudemycin) is used to induce skipping of exons 4, 10, and 11 of the MDM2 minigene. Similarly, cell type-specific or disease state alternative splicing events may be used as minigenes inserted into the coding sequence of a target gene.

[0089] When the chimeric transactivator minigene or chimeric target gene minigene has a minigene inserted at the 5' end of the transactivator or target gene coding sequence, the optionally included exon may contain the necessary translation initiation regulatory sequences. In addition, a cleavable peptide may be located between the minigene sequence and the transactivator or target gene, as described above.

[0090] It should be noted that other types of alternative splicing events may also be used in the proposed system. For example, one skilled in the art will recognize that alternative 3' or alternative 5' splice sites can be engineered to serve the same purpose as alternatively skipped or alternatively included exons. Similarly, retained intron splicing events can be engineered accordingly.

[0091] I. Target Genes for Alternative Splicing Regulation A. Inhibitory RNA "RNA interference (RNAi)" is the process of sequence-specific post-transcriptional gene silencing initiated by siRNA. During RNAi, siRNA induces the degradation of target mRNA, resulting in sequence-specific inhibition of gene expression. Examples of genes whose expression can be inhibited using the expression system of the present disclosure include, but are not limited to, HTT (for Huntington's disease), SCA (for spinocerebellar ataxia (types 1, 2, 3, 6, 7)), FXTAS (for fragile X ataxia syndrome), and FMRP (for fragile X).

[0092] "Inhibitory RNA", "RNAi", "small interfering RNA" or "short interfering RNA" or "siRNA" molecule, "short hairpin RNA" or "shRNA" molecule, or "miRNA" is an RNA duplex of nucleotides targeted to a nucleic acid sequence of interest. As used herein, the term "siRNA" is a general term that encompasses a subset of shRNA and miRNA. "RNA duplex" refers to the structure formed by the complementary pairing between two regions of an RNA molecule. The nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the targeted gene, so that the siRNA is "targeted" to that gene. In certain embodiments, the siRNA is targeted to the sequence encoding Huntington. In some embodiments, the length of the duplex of the siRNA is less than 30 base pairs. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 base pairs in length. In some embodiments, the length of the duplex is 19-25 base pairs long. In certain embodiments, the length of the duplex is 19 base pairs long or 21 base pairs long. The RNA duplex portion of the siRNA can be part of a hairpin structure. The hairpin structure contains, in addition to the duplex portion, a loop portion located between the two sequences forming the duplex. The length of the loop can vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides long. In certain embodiments, the loop is 18 nucleotides long. The hairpin structure can also contain a 3' overhang portion and / or a 5' overhang portion. In some embodiments, the overhang is a 3' overhang and / or a 5' overhang that is 0, 1, 2, 3, 4 or 5 nucleotides long.

[0093] shRNAs are composed of stem-loop structures designed to contain a 5' flanking region, an siRNA region segment, a loop region, a 3' siRNA region, and a 3' flanking region. Most RNAi expression strategies have utilized short hairpin RNAs (shRNAs) driven by strong polIII promoters. Many shRNAs have shown effective knockdown of target sequences both in vitro and in vivo, but some shRNAs that show effective knockdown of target genes have also been found to be toxic in vivo.

[0094] miRNA is a small cellular RNA (about 22nt) that is processed from precursor stem-loop transcript.Known miRNA stem-loop can be modified to contain RNAi sequence specific to gene of interest.miRNA molecule may be preferred to shRNA molecule because miRNA is endogenously expressed.Therefore, miRNA molecule is less likely to induce dsRNA-responsive interferon pathway, is more efficiently processed than shRNA, and has been shown to silence 80% more effectively than shRNA.

[0095] An alternative approach that has been discovered recently is the use of artificial miRNA (pri-miRNA scaffold shuttles siRNA sequence) as RNAi vector.Artificial miRNA is more similar to endogenous RNAi substrates in nature and is more suitable for Pol-II transcription (e.g., allows tissue-specific expression of RNAi) and polycistronic strategy (e.g., allows delivery of multiple siRNA sequences).See U.S. Patent No. 10,093,927, which is incorporated herein by reference.

[0096] The transcription unit of an "shRNA" consists of a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides. shRNAs are exported from the nucleus by exportin-5 and processed by Dicer in the cytoplasm to generate functional siRNAs. The stem-loop of an "miRNA" consists of a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides, which are typically expressed as part of a larger primary transcript (pri-miRNA), which is excised by the Drosha-DGCR8 complex to generate an intermediate known as a pre-miRNA, which is then exported from the nucleus by exportin-5 and processed by Dicer in the cytoplasm to generate functional siRNAs. As used interchangeably herein, "artificial miRNA" or "artificial miRNA shuttle vector" refers to a primary miRNA transcript in which the region of the double-stranded stem-loop (at least about 9-20 nucleotides) excised by Drosha and Dicer processing is replaced with an siRNA sequence for the target gene while retaining the structural elements in the stem-loop required for effective Drosha processing. The term "artificial" comes from the fact that the flanking sequences (about 35 nucleotides upstream and about 40 nucleotides downstream) are derived from restriction enzyme sites within the multiple cloning site of the siRNA. As used herein, the term "miRNA" encompasses both naturally occurring miRNA sequences and artificially created miRNA shuttle vectors.

[0097] siRNA can be encoded by nucleic acid sequence, and the nucleic acid sequence can also include a promoter.The nucleic acid sequence can also include a polyadenylation signal.In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a sequence of six T's.

[0098] In the design of RNAi, there are several factors that need to be considered, such as the nature of the siRNA, the permanence of the silencing effect and the choice of delivery system. To produce the RNAi effect, the siRNA introduced into the organism will typically contain exonic sequences. Furthermore, since the RNAi process depends on homology, the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. Preferably, the siRNA exhibits greater than 80%, 85%, 90%, 95% or 98% identity, or even 100% identity, between the sequence of the siRNA and the gene to be inhibited. Sequences with less than about 80% identity to the target gene are much less effective. Thus, the higher the homology between the siRNA and the gene to be inhibited, the less likely the expression of unrelated genes will be affected.

[0099] In addition, the size of the siRNA is also an important consideration. In some embodiments, the present invention relates to siRNA molecules that contain at least about 19-25 nucleotides and can regulate gene expression. In the context of the present invention, the siRNA is preferably less than 500, 200, 100, 50 or 25 nucleotides in length. More preferably, the siRNA is about 19 nucleotides to about 25 nucleotides in length.

[0100] By siRNA target is generally meant a polynucleotide that comprises a region that codes for a polypeptide, or a polynucleotide that comprises a region that regulates the replication, transcription or translation or other processes important for the expression of a polypeptide, or a polynucleotide that comprises both a region that codes for a polypeptide and a region that is functionally linked thereto and regulates its expression.Any gene that is expressed in cells can be targeted.Preferably, the target gene is involved or associated with the progression of a cellular activity that is important for disease or that is of particular interest for research.

[0101] B. CRISPR Systems Gene editing is a technique that allows targeted genetic modification in living cells. In recent years, the implementation of on-demand gene editing utilizing the bacterial CRISPR immune system has revolutionized the way scientists approach genome editing. The Cas9 protein of the CRISPR system, an RNA-guided DNA endonuclease, can be relatively easily engineered to target new sites by altering its guide RNA sequence. With this discovery, sequence-specific gene editing has become functionally enabled.

[0102] In general, "CRISPR system" refers collectively to transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, such as sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and, in the case of endogenous CRISPR systems, partial direct repeats processed by tracrRNA), guide sequences (also referred to as "spacers" in the case of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus. Examples of genes whose expression may be inhibited or whose sequences may be edited using the CRISPR expression system of the present disclosure include, but are not limited to, HTT (for Huntington's disease), SCA (for Spinocerebellar Ataxia (Types 1, 2, 3, 6, 7)), FXTAS (for Fragile X Ataxia Syndrome), and FMRP (for Fragile X).

[0103] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) with nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a Type I, Type II, or Type III CRISPR system, for example, from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes.

[0104] The CRISPR system can induce a double stranded break (DSB) at the target site and subsequent scission, as discussed herein. In another embodiment, a Cas9 variant considered a "nickase" is used to nick one strand at the target site. A pair of nickases can be used, each directed by a pair of different gRNAs that target sequences in such a way that a 5' overhang is introduced when the nicks are introduced simultaneously, for example to improve specificity. In another embodiment, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor (e.g., KRAB) or transcriptional activator, to affect gene expression. Alternatively, the CRISPR system with catalytically inactive Cas9 further comprises a transcriptional repressor or transcriptional activator fused to a ribosome binding protein.

[0105] In some aspects, Cas nuclease and gRNA (comprising a fusion of target sequence-specific crRNA and invariant tracrRNA) are introduced into cells. In general, the target site at the 5' end of gRNA targets Cas nuclease to the target site, e.g., gene, using complementary base pairing. The target site can be selected based on its location, immediately 5' to a protospacer adjacent motif (PAM) sequence, e.g., typically NGG or NAG. In this regard, gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11 or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the site of the target sequence. Generally, "target sequence" generally refers to the sequence that guide sequence is designed to have complementarity with, and the hybridization between target sequence and guide sequence promotes the formation of CRISPR complex.Complete complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of CRISPR complex.

[0106] The target sequence may comprise any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, such as in a cell organelle. In general, the sequence or template that can be used for recombination into a targeted locus that comprises a target sequence is called an "editing template" or an "editing polynucleotide" or an "editing sequence". In some aspects, the exogenous template polynucleotide may be called an editing template. In some aspects, the recombination is a homologous recombination.

[0107] Typically, in the case of endogenous CRISPR systems, the formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs or more from the target sequence). The tracr sequence, which may include or consist of all or a portion of the wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85 nucleotides or more of the wild-type tracr sequence), may also form part of a CRISPR complex, for example, by hybridization to all or a portion of the tracr mate sequence operably linked to the guide sequence along at least a portion of the tracr sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, e.g., at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity over the entire length of the tracr mate sequence when optimally aligned.

[0108] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into the cell, so that the expression of those elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. Components can also be delivered to the cell as proteins and / or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence could each be operably linked to separate regulatory elements on separate vectors. The Cas enzyme can be a target gene that is under the control of a regulated alternative splicing event as disclosed herein as a chimeric target gene minigene or as a target gene for a chimeric minigene transactivator. The gRNA can be under the control of a constitutive promoter.

[0109] Alternatively, two or more elements expressed from the same or different regulatory elements may be combined in one vector, providing any components of the CRISPR system that one or more additional vectors are not included in the first vector. A vector may include one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. By using multiple different guide sequences, a single expression construct may be used to target CRISPR activity to multiple corresponding target sequences in a cell.

[0110] The vector can include regulatory elements operably linked to an enzyme coding sequence that encodes a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof. These enzymes are known, for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0111] The CRISPR enzyme can be Cas9 (e.g., from Streptococcus pyogenes or S. pneumonia). The CRISPR enzyme can direct cleavage of one or both strands at the location of the target sequence, e.g., within the target sequence and / or within the complementary strand of the target sequence. The vector can encode a CRISPR enzyme that is mutated such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence, as compared to the corresponding wild-type enzyme. For example, an aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (one strand cleavage). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence, e.g., two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination allows both strands to be nicked and used to induce NHEJ or HDR.

[0112] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be of or derived from a particular organism, such as a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon of the native sequence with a codon that is more or most frequently used in the host cell's genes while maintaining the native amino acid sequence, in order to enhance expression in the host cell of interest. Different species exhibit a certain bias for certain codons of a particular amino acid. Codon bias (the difference in codon usage between organisms) is often correlated with the efficiency of messenger RNA (mRNA) translation, and is believed to depend, among other things, on the properties of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of a selected tRNA in a cell is generally a reflection of the codon that is most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0113] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more when optimally aligned using a suitable alignment algorithm.

[0114] Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn) and Maq (available at maq.sourceforge.net).

[0115] CRISPR enzymes may be part of a fusion protein that includes one or more heterologous protein domains. CRISPR enzyme fusion proteins may include any additional protein sequences and, optionally, a linker sequence between any two domains. Examples of protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription termination factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent protein including blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences that code for proteins or fragments of such proteins that bind to DNA molecules or other cellular molecules, such as, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can form part of fusion proteins that contain CRISPR enzymes are described in US 20110059502, which is incorporated herein by reference.

[0116] C. Therapeutic Proteins Some embodiments relate to the expression of recombinant proteins and polypeptides. Examples of proteins that can be expressed using the expression systems of the present disclosure include STXBP1 (also known as Munc18-1; for STXBP1 deficiency, a form of neonatal epilepsy, a form of developmental delay), SCN1a (for Dravet syndrome, also known as hereditary epileptic encephalopathy, also known as severe myoclonic epilepsy in infancy (SMEI); mutations in Nav1.1); SCN1b (mutations in Nav1.1 β subunit); SCN2b (for familial atrial fibrillation; β2 subunit of the type II voltage-gated sodium channel); KCNA1 (for overt hereditary paroxysmal ataxia; for muscle spasms with or without ataxia). rigidity);KCNQ2 (KCNQ2-related epilepsy);GABRB3 (early onset epilepsy; beta 3 subunit of GABAA receptor);CACNA1A (for familial ataxia and hemiplegic migraine; transmembrane pore-forming subunit of P / Q-type voltage-gated calcium channel);CHRNA2 (for autosomal overt nocturnal frontal lobe epilepsy; alpha subunit of neuronal nicotinic cholinergic receptor (nAChR);KCNT1 (for autosomal overt nocturnal frontal lobe epilepsy (ADNFLE) and malignant focal moving partial seizures of infancy (MMPSI); sodium-activated potassium channel);SCN8A (for epilepsy and neurodevelopmental disorders; Nav1.6 deficiency, voltage-gated sodium channel); CHRNA4-α subunit (for autosomal overt nocturnal frontal lobe epilepsy; mutation in the α subunit of the nicotinic acetylcholine receptor); CHRNB2-b2 subunit (for autosomal overt nocturnal frontal lobe epilepsy; mutation in the α subunit of the nicotinic acetylcholine receptor); ARX (for Otohara syndrome, poly Ala expansion in the ARX gene); MECP1 (for Rett syndrome); FMRP (for Fragile X); and CLN3 (for CLN disease, also known as juvenile Batten disease, also known as JNCL).Other examples of proteins that may be expressed using the expression system of the present disclosure can be found in Lindy et al. (2018) and Heyne et al. (2018), each of which is incorporated by reference in its entirety.

[0117] In some aspects, proteins or polypeptides may be modified to increase serum stability. Thus, when the present application refers to a function or activity of a "modified protein" or "modified polypeptide," it will be understood by those skilled in the art that it encompasses proteins or polypeptides that have additional advantages over, for example, unmodified proteins or polypeptides. It is specifically contemplated that embodiments relating to "modified proteins" can also be implemented with respect to "modified polypeptides," and vice versa.

[0118] Recombinant proteins may have deletions and / or substitutions of amino acids. Thus, proteins with deletions, proteins with substitutions, and proteins with deletions and substitutions are modified proteins. In some embodiments, these proteins may further include inserted or added amino acids, such as fusion proteins or proteins with linkers. A "modified deleted protein" may lack one or more residues of the native protein but may have the specificity and / or activity of the native protein. A "modified deleted protein" may also have reduced immunogenicity or antigenicity. An example of a modified deleted protein is one in which amino acid residues are deleted from at least one antigenic region, i.e., from a region of the protein determined to be antigenic in a particular organism, e.g., in a type of organism to which the modified protein may be administered.

[0119] Substitution or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein and can be designed to modulate one or more properties of the polypeptide, particularly its effector functions and / or bioavailability. Substitutions may or may not be conservative, i.e., one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, an alanine to serine change, an arginine to lysine change, an asparagine to glutamine or histidine change, an aspartic acid to glutamic acid change, a cysteine ​​to serine change, a glutamine to asparagine change, a glutamic acid to aspartic acid change, a glycine to proline change, a histidine to asparagine or glutamine change, an isoleucine to leucine or valine change, a leucine to valine or isoleucine change, a lysine to arginine change, a methionine to leucine or isoleucine change, a phenylalanine to tyrosine, leucine or methionine change, a serine to threonine change, a threonine to serine change, a tryptophan to tyrosine change, a tyrosine to tryptophan or phenylalanine change, and a valine to isoleucine or leucine change.

[0120] In addition to deletions or substitutions, modified proteins may have the insertion of residues. This typically involves the addition of at least one residue in the polypeptide. This may include the insertion of a targeting peptide or targeting polypeptide or simply the insertion of a single residue. Terminal additions, called fusion proteins, are described below.

[0121] The term "biologically functional equivalent" is well understood in the art and is further described herein. Thus, sequences in which about 70% to about 80%, or about 81% to about 90%, or even about 91% to about 99% of the amino acids are identical or functionally equivalent to those of a reference polypeptide are encompassed, so long as the biological activity of the protein is maintained. A recombinant protein may, in certain aspects, be a biologically functional equivalent to the corresponding native protein.

[0122] It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, and still be essentially as described in one of the sequences disclosed herein, so long as the sequence meets the above criteria, including, where protein expression is concerned, maintenance of biological protein activity. The addition of terminal sequences is particularly applicable to nucleic acid sequences which may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region, or may include various internal sequences, i.e., introns, that are known to be present within genes.

[0123] As used herein, a protein or peptide generally refers to, but is not limited to, a protein of more than about 200 amino acids, up to the full-length sequence translated from a gene; a polypeptide of more than about 100 amino acids; and / or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0124] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art. In certain embodiments, the residues of a protein or peptide are contiguous, without any non-amino acid sequence interrupting the sequence of amino acid residues. In other embodiments, the sequence may include one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0125] Thus, the term "protein or peptide" encompasses amino acid sequences that include at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.

[0126] Certain aspects of the present invention relate to fusion proteins. These molecules may have a therapeutic protein linked to a heterologous domain at the N-terminus or C-terminus. For example, fusions may also use leader sequences from other species to allow recombinant expression of the protein in a heterologous host. Other useful fusions include the addition of a protein affinity tag, preferably cleavable, such as a serum albumin affinity tag or six histidine residues, or an immunologically active domain, such as an antibody epitope, to facilitate purification of the protein. Non-limiting examples of affinity tags include polyhistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST).

[0127] Methods for making fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by de novo synthesis of an entire fusion protein, or by attachment of a DNA sequence encoding a heterologous domain followed by expression of the intact fusion protein.

[0128] The production of fusion proteins that restore the functional activity of the parent protein can be facilitated by ligating the genes with a bridging DNA segment that encodes a peptide linker that is joined between the tandemly linked polypeptides, the linker being of sufficient length to allow proper folding of the resulting fusion protein.

[0129] II. Splicing Modifiers An exemplary splice modifier is LMI070 (Spinraza™, Novartis 31 ), which can penetrate the blood-brain barrier and has the following structure: TIFF2025060942000004.tif19128

[0130] Examples of alternative splicing events in which novel exons are included only in the presence of LMI070 and can be used to control gene expression in the disclosed system include SF3B3 (chr16:70,526,657-70,529,199), BENC1 (chr17:42,810,759-42,811,797), GXYLT1 (chr12:42,087,786-42,097,614), SKP1 (chr5:134,173,809-134,177,053), C12orf4 (chr12:134,173,809-134,177,053), and CYP2 (chr12:134,173,809-134,177,053). :4,536,017~4,538,508), SSBP1(chr7:141,739,167~141,742,229), RARS(chr5:168,517,815~168,519,190), PDXDC2P(chr16:70,030,98 8~70,031,968), STRADB(chr2:201,469,953~201,473,076), WNK1(chr12:894,562~896,732), WDR27(chr6:169,660,663~169,662,424), CI P2A(chr3:108,565,355~108,566,638), IFT57(chr3:108,191,521~108,206,696), WDR27(chr6:169,660,649~169,662,458), HTT(chr4:3 ,212,555~3,214,145), SKA2(chr17:59,112,228~59,119,514), EVC(chr4:5,733,318~5,741,822), DYRK1A(chr21:37,420,144~37,473,05 6), GNAQ (chr9:77,814,652-77,923,557), ZMYM6 (chr1:35,019,257-35,020,472), CYB5B (chr16:69,448,031-69,459,160), MMS22L (chr6:97,186,342-97,229,533), MEMO1 (chr2:31,883,262-31,892,301) and PNISR (chr6:99,416,278-99,425,413).Examples of alternative splicing events that have enhanced inclusion of novel exons in the presence of LMI070 and can be used to control gene expression in the disclosed system include CACNA2D1 (chr7:82,066,406-82,084,958), SSBP1 (chr7:141,739,083-141,742,248), DDX42 (chr17:63,805,048-63,806,672), ASAP1 (chr8:130,159,817-130,167,6 88), DUXAP10 (chr14:19,294,564~19,307,199), AVL9 (chr7:32,558,783~32,570,372), DYRK1A (chr21:37,419,920~37,472,960), FAM3A(chrX:154,512,311~154,512,939), FHOD3(chr18:36,740,620~36,742,886), TBCA(chr5:77,707,994~77,777,000), MZT1(c hr13:72,718,939~72,727,611), LINC01296(chr14:19,092,877~19,096,652), SF3B3(chr16:70,541,627~70,544,553), SAFB(chr 19:5,654,060~5,654,457), GCFC2(chr2:75,702,163~75,706,652), MRPL45(chr17:38,306,450~38,319,088), SPIDR(chr8:47,26 These include, but are not limited to, DUXAP8 (chr22:15,815,315-15,828,713), PDXDC1 (chr16:15,008,772-15,009,763), MAN1A2 (chr1:117,442,104-117,461,030), RAF1 (chr3:12,600,376-12,604,350) and ERGIC3 (chr20:35,548,787-35,554,452). For the above list, each genomic location includes the upstream and downstream exons and intervening intronic sequences targeted by LMI070.

[0131] Also included are analogs of splice modifiers such as LMI070 that can be used similarly, such as, for example, 6-(naphthalen-2-yl)-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine, 6-(benzo[b]thio-phen-2-yl)-N-methyl-N-(2,2,6,6-tetra-methylpiperidin-4-yl)pyridazin-3-amine, 2-(6-(2,2,6,6-tetramethylpiperidin-4-ylamino)-pyridazin-3-yl)phenol, 2-(6-( ... 2-(6-(methyl-(2,2,6,6-tetramethylpiperidin-4-yl)amino)-pyridazin-3-yl)benzo[b]-thiophene-5-carbonitrile, 6-(quinolin-3-yl)-N-(2,2,6,6-tetramethyl-piperidin-4-yl)pyridazin-3-amine, 3-(benzo[b]-thiophen-2-yl)-6-(2,2,6,6-tetramethylpiperidin-4-yloxy)pyridazine, 2-(6-(methyl-(2,2,6,6-tetramethylpiperidin-4-yl)amino)-pyridazin-3-yl)phenol, 6-( 6-(methyl-(2,2,6,6-tetramethylpiperidin-4-yl)amino)-pyridazin-3-yl)naphthalen-2-ol, 6-(benzo[b]-thiophen-2-yl)-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine, 7-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)isoquinoline, 6-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)isoquinoline, N-methyl-6-(quinol N-methyl-6-(quinolin-6-yl)-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine, 6-(isoquinolin-7-yl)-N-methyl-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine, 6-(isoquinolin-6-yl)-N-methyl-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine, 6-(imidazo[1,2-a]pyridin-6-yl-pyridazin-3-yl)-methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amine, methyl-[6-(6-phenyl-pyridin-3-yl)-pyridazin-3-yl]-(2,2,6,6-tetramethyl-piperidin-4-yl)-amine, methyl-[6-(6-pyrrol-1-yl-pyridin-3-yl)-pyridazin-3-yl]-(2,2,6,6-tetramethyl-piperidin-4-yl)-amine, methyl-[6-(6-pyrazole-1-yl-pyridin-3-yl)-pyridazin-3-yl]-(2,2,6,6-tetramethyl-piperidin-4-yl)-amine, methyl-(6-quinoxalin-2-yl-pyridazin-3-yl)-(2,2,6,6-tetramethyl-piperidin-4-yl)-amine, methyl-(6-quinolin-3-yl-pyridazin-3-yl)-(2,2,6,6-tetramethyl-piperidin-4-yl)-amine, N-methyl-6-(phthalazin-6-yl)-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine, 6-(benzo[c][1,2,5]oxa-diazole-5- 6-(benzo[d]thiazol-5-yl)-N-(2,2,6,6-tetramethyl-piperidin-4-yl)pyridazin-3-amine, 6-(2-methylbenzo-[d]oxazol-6-yl)-N-(2,2,6,6-tetramethyl-piperidin-4-yl)pyridazin-3-amine, 3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalen-2-ol, 5-chloro-2-(6 -(Methyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 3-(6-(2,2,6,6-tetramethylpiperidin-4-ylamino)pyridazin-3-yl)naphthalen-2-ol, 5-chloro-2-(6-(1,2,2,6,6-pentamethylpiperidin-4-ylamino)pyridazin-3-yl)phenol, 4-hydroxy-3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)benzonitrile, 3-[6-(2,2,6,6-Tetramethyl-piperidin-4-yloxy)-pyridazin-3-yl]-naphthalen-2-ol, 2-{6-[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]-pyridazin-3-yl}-4-trifluoromethyl-phenol, 2-fluoro-6-{6-[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]-pyridazin-3-yl}-phenol, 3,5-dimethoxy-2-{6-[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]-pyridazin-3-yl}-phenol, 4,5-dimethoxy-2-{6-[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]-pyridazin-3-yl}-phenol, 5-methoxy-2-{6-[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]pyridazin-3-yl}-phenol, 4,5-difluoro-2-{6-[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]-pyridazin-3-yl}-phenol yl}-phenol, 5-fluoro-2-{6-[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]-pyridazin-3-yl}-phenol, 3-hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)benzonitrile, 1-allyl-6-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalen-2-ol, 6-(benzo[b]thiophene-2- N-allyl-3-hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)benzamide, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1H-pyrazol-1-yl)phenol, 5-(5-methyl-oxazol-2-yl)-2-{6-[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]pyridazin-3-yl}-phenol, 5-(4-hydroxymethyl)-1H-pyrazol-1-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-(1H-imidazol-1-yl)-2-(6-(methyl(2,2,6,6-tetramethyl-piperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-(4-amino-1H-pyrazol-1-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol pyridazin-3-yl)phenol, 5-(4-amino-1H-pyrazol-1-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-(3-amino-pyrazol-1-yl)-2-{6-[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]pyridazin-3-yl}-phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1-(2-morpholino-ethyl)-1H-pyrazol-4-yl)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1-methyl-1H-pyrazol-4-yl)phenol, 5-(5-amino-1H-pyrazol-1-yl)-2-(6-(methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)amino)pyridazin-3-yl)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-4-(1H-pyrazol-1-yl)phenol, 2-{6-[(2 -hydroxy-ethyl)-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]pyridazin-3-yl}-5-pyrazol-1-yl-phenol, 2-(6-(piperidin-4-yloxy)pyridazin-3-yl)-5-(1H-pyrazol-1-yl)phenol, 2-(6-(((2S,4R,6R)-2,6-dimethylpiperidin-4-yl)oxy)pyridazin-3-yl)-5-(1H-pyrazol-1-yl)phenol, 2-(6-((-2,6-dimethylpiperidin-4-yl)oxy)pyridazin-3-yl)-5-(1H-pyrazol-1-yl)phenol, 2-(6-((-2,6-dimethylpiperidin-4-yl)oxy)pyridazin-3-yl)-5-(1H-pyrazol-1-yl)phenol, 5-(1H-pyrazol-1-yl)-2-(6-(pyrrolidin-3-yloxy)pyridazin-3-yl)phenol, 2-(6-((-2-methylpiperidin-4-yl)oxy)pyridazin-3-yl)-5-(1H-pyrazol-1-yl)phenol, (S)-5-(1H-pyrazol-1-yl)-2-(6-(pyrrolidine -3-ylmethoxy)pyridazin-3-yl)phenol, (R)-5-(1H-pyrazol-1-yl)-2-(6-(pyrrolidin-3-ylmethoxy)pyridazin-3-yl)phenol, 2-(6-((3-fluoropiperidin-4-yl)oxy)pyridazin-3-yl)-5-(1H-pyrazol-1-yl)-phenol, 2-[6-(1,2,2,6,6-pentamethyl-piperidin-4-yloxy)-pyridazin-3-yl]-5-pyrazol-1-yl-phenol, 5-pyrazol-1-yl-2-[6-(2,2,6,6-Tetramethyl-piperidin-4-yloxy)-pyridazin-3-yl]-phenol, 5-(1H-pyrazol-4-yl)-2-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)phenol, 2-(6-piperazin-1-yl-pyridazin-3-yl)-5-pyrazol-1-yl-phenol, 3-[6-(azetidin-3-ylamino)-pyridazin-3-yl]-naphthalen-2-ol, 2-[6-(azetidin-3-ylamino)-pyridazin-3-yl] -5-pyrazol-1-yl-phenol, 2-[6-(3,5-dimethyl-piperazin-1-yl)-pyridazin-3-yl]-5-pyrazol-1-yl-phenol, 2-[6-(7-methyl-2,7-diaza-spiro[4.4]non-2-yl)-pyridazin-3-yl]-5-pyrazol-1-yl-phenol, 2-(6-[1,4]diazepan-1-yl-pyridazin-3-yl)-5-pyrazol-1-yl-phenol, 2-{6-[4-(2-hydroxy-ethyl)-piperazin-1-yl]-pyridazine -3-yl}-5-pyrazol-1-yl-phenol, 2-[6-(3,6-diaza-bicyclo[3.2.1]oct-3-yl)-pyridazin-3-yl]-5-pyrazol-1-yl-phenol, 2-[6-(2,7-diaza-spiro[3.5]non-7-yl)-pyridazin-3-yl]-5-pyrazol-1-yl-phenol, 2-[6-(3-hydroxy-methyl-piperazin-1-yl)-pyridazin-3-yl]-5-pyrazol-1-yl-phenol, 2-[6-(1,7-diaza-spiro[4.4] non-7-yl)-pyridazin-3-yl]-5-pyrazol-1-yl-phenol, 2-[6-(4-amino-4-methyl-piperidin-1-yl)-pyridazin-3-yl]-5-pyrazol-1-yl-phenol, 2-[6-(3-dimethyl-amino-piperidin-1-yl)-pyridazin-3-yl]-5-pyrazol-1-yl-phenol, 2-[6-(1,2,2,6,6-pentamethyl-piperidin-4-ylamino)-pyridazin-3-yl]-5-pyrazol-1-yl-phenol, 2-[6-(3,3-Dimethyl-piperazin-1-yl)-pyridazin-3-yl]-5-pyrazol-1-yl-phenol, 2-(6-(7-(2-hydroxyethyl)-2,7-diazaspiro[4.4]-nonan-2-yl)pyridazin-3-yl)-5-(1H-pyrazol-1-yl)phenol, 2-(6-((3aR,6aS)-hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridazin-3-yl)-5-(1H-pyrazol-1-yl)phenol, 3-(6-(piperazin-1-yl)pyridazin-3-yl)naphthalene -2,7-diol, 5-pyrazol-1-yl-2-[6-(1,2,3,6-tetrahydro-pyridin-4-yl)-pyridazin-3-yl]-phenol, 2-(6-piperidin-4-yl-pyridazin-3-yl)-5-pyrazol-1-yl-phenol, 3-(6-(1,2,3,6-tetrahydropyridin-4-yl)pyridazin-3-yl)naphthalene-2-ol, 3-(6-(1,2,3,6-tetrahydropyridin-4-yl)pyridazin-3-yl)naphthalene-2,7-diol, 3-(6-(2,2,6,6-tetrahydropyridin-4-yl)pyridazin-3-yl)naphthalene-2,7-diol, 3-(6-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)pyridazin-3-yl)naphthalene-2,7-diol, 3-(6-(piperidin-4-yl)pyridazin-3-yl)naphthalene-2,7-diol, 3-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)naphthalene-2,7-diol, 3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)naphthalene-2,7-diol, 3-(6-((2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalene-2,7-diol, [3-(7-hydroxy-6-{6-[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)amino]-pyridazin-3-yl}-naphthalene-2-yloxy)-propyl]-carbamic acid tert-butyl ester, 7-(3-amino-propoxy)-3-{6-[methyl-(2,2,6,6-Tetramethyl-piperidin-4-yl)-amino]-pyridazin-3-yl}-naphthalen-2-ol, N-[3-(7-hydroxy-6-{6[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]-pyridazin-3-yl}-naphthalen-2-yloxy)-propyl]-acetamide, 7-(3-hydroxypropoxy)-3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalen-2-ol, 7-(3-methoxypropoxy) -3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalen-2-ol, 7-(2-morpholinoethoxy)-3-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)naphthalen-2-ol, 3-(6-(piperidin-4-ylmethyl)pyridazin-3-yl)naphthalen-2-ol, 5-(1H-pyrazol-1-yl)-2-(6-((2,2,6,6-tetramethylpiperidin-4-yl)methyl)pyridazine -3-yl)phenol, 3-methoxy-2-(6-(methyl(2,2,6-trimethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(5-methyloxazol-2-yl)phenol, 2-(6-((6S)-6-((S)-1-hydroxyethyl)-2,2-dimethylpiperidin-4-yloxy)pyridazin-3-yl)-5-(1H-pyrazol-1-yl)phenol, 7-hydroxy-6-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-2 -naphthonitrile, 3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-7-(piperidin-1-ylmethyl)naphthalene-2-ol, 3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-7-(pyrrolidin-1-ylmethyl)naphthalene-2-ol, 1-bromo-6-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalene-2,7-diol, 1-chloro-6-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalene-2,7-diol, 7-methoxy-3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalene-2-ol, 7-methoxy-3-(6-(methyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalene-2-ol, 7-(3,6-dihydro-2H-pyran-4-yl)-3-(6-(methyl(2, 2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalen-2-ol, 3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-7-(tetrahydro-2H-pyran-4-yl)naphthalen-2-ol, 7-(difluoromethyl)-3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalen-2-ol, 7-((4-hydroxy-2-methylbutan-2-yl)oxy)-3-(6-(methyl 3-Methoxy-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalen-2-ol, 7-(3-hydroxy-3-methylbutoxy)-3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalen-2-ol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1H-pyrazol-4-yl)benzene-1,3-diol, 3-Methoxy-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalen-2-ol, 5-(1H-pyrazol-4-yl)-2-(6-((2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-3-(trifluoromethoxy)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1-methyl-1H-pyrazol-4-yl)-3-(trifluoromethoxy)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1H-pyrazol-4-yl)-3-(trifluoromethoxy)phenol, 4-(3-hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(trifluoromethoxy)phenyl)-1-methylpyridin-2(1H)-one, 3-methoxy-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1-methyl-1H-pyrazol-4-yl) pyrazol-4-yl)phenol, 3-methoxy-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)phenol, 3-methoxy-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(pyridin-3-yl)phenol, 5-(1-cyclopentyl-1H-pyrazol-4-yl)-3-methoxy-2-(6-(methyl(2,2,6 ,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 3',5-dimethoxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-[1,1'-biphenyl]-3-ol, 3-(benzyloxy)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(5-methyloxazol-2-yl)phenol, 3-ethoxy-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)- 3-(cyclopropylmethoxy)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)-pyridazin-3-yl)-5-(5-methyloxazol-2-yl)phenol, 2-methyl-5-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-1H-benzo[d]imidazol-6-ol, 5-chloro-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-(1H-pyrazol-1-yl)-2-(6-((2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 3-hydroxy-4-(6-((2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)benzonitrile, 2-(6-((2,2-dimethylpiperidin-4-yl)oxy)pyridazin-3-yl)-5-(1H-pyrazol-1-yl)phenol, 2-(6-(methyl( 2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-4-(1H-pyrazol-4-yl)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-4-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-4-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-3-yl)furan phenol, 4-(1H-indol-2-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 4-(cyclopent-1-en-1-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-4-(1H-pyrazol-3-yl)phenol, 4-(4-hydroxy-3-(6-(methyl 4-(4-hydroxy-3-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)phenyl)pyridin-2-ol, 4-(4-hydroxy-3-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)phenyl)-1-methylpyridin-2(1H)-one, 4-(4-hydroxy-3-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)phenyl)pyridin-2-ol, 5-(1H-indazol-7-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 4-chloro-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1H-pyrazol-4-yl)phenol, 4-fluoro-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1H-pyrazol-4-yl)phenol, 5-fluoro-4-(1H-imidazol-4-yl)-2-(6-(methyl(2,2,6 ,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-fluoro-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-4-(1H-pyrazol-4-yl)phenol, 5-fluoro-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-4-(1H-pyrazol-5-yl)phenol, 6-hydroxy-5-(6-(methyl(2,2,6,6-tetramethylpiperidine-4-yl)amino)pyridazin-3-yl)-4-(1H-pyrazol-5-yl)phenol -yl)amino)pyridazin-3-yl)-2,3-dihydro-1H-inden-1-one, 6-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-1,4-dihydroindeno[1,2-c]pyrazol-7-ol, 6-hydroxy-5-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-2,3-dihydro-1H-inden-1-one oxime hydrochloride, 5-(6-(methyl(2,2,6,6-tetramethylpiperidine- 4-yl)amino)pyridazin-3-yl)-2,3-dihydro-1H-indene-1,6-diol, 2-amino-6-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-8H-indeno[1,2-d]thiazol-5-ol hydrochloride, 9-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5,6-dihydroimidazo[5,1-a]isoquinolin-8-ol hydrochloride, 4-hydroxy-3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-N-((1-methyl-1H-pyrazol-4-yl)methyl)benzamide, 4-(4-(hydroxymethyl)-1H-pyrazol-1-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-(1H-pyrazol-4-yl)-2-(6-((2,2,6,6-tetramethylpiperidin-4-yl)methyl)pyridazin-3-yl)phenol, 6-(3-(benzyloxy)isoquinoline-6 -yl)-N-methyl-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine, 6-(1-(benzyloxy)isoquinolin-7-yl)-N-methyl-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine, 3-fluoro-5-(2-methoxypyridin-4-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol hydrochloride, 4-(3-fluoro-5-hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol hydrochloride, 4-(3-fluoro-5-hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenyl)pyridin-2(1H)-one hydrochloride, 5-(3-fluoro-5-hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenyl)-1-methylpyridin-2(1H)-one hydrochloride, 3-fluoro-5 -(1H-pyrazol-4-yl)-2-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)phenol hydrochloride, 5-chloro-3-fluoro-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol hydrochloride, 3-fluoro-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1H-pyrazol-4-yl)phenol hydrochloride, 3-fluoro-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1-methyl-1H-pyrazol-4-yl)phenol hydrochloride, 5-(5-methoxypyridin-3-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-(3-hydroxy-4-(6-methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenyl)pyridin-2-ol, 4-(3-hydroxy-4-(6-methyl( 2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenyl)pyridin-2-ol, 5-(6-methoxypyridin-3-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-(3-hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenyl)-3-(trifluoromethyl)pyridin-2-ol, 5-(3-hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenyl)- 4-(3-hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenyl)-1-methylpyridin-2(1H)-one, 5-(2-methoxypyridin-4-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 4-(3-hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenyl)-1-methylpyridin-2(1H)-one, -(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)phenyl)pyridin-2-ol, 5-(6-(dimethylamino)pyridin-3-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 4-(3-hydroxy-4-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)phenyl)-1-methylpyridin-2(1H)-one, 2-(6-(methyl(2,2,6,6-Tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(pyrimidin-5-yl)phenol, 5-(3-hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenyl)pyridin-3-ol, 1-cyclopropyl-4-(3-hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenyl)pyridin-2(1H)-one, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1,2,3,6-tetrahydropyridin-4-yl)phenyl 5-(cyclopent-1-en-1-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-(3,6-dihydro-2H-pyran-4-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-(imidazo[1,5-a]pyridin-7-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-(imidazo[1,5-a]pyridin-7-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, Midazo[1,2-a]pyridin-7-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(2-methylpyridin-4-yl)phenol, 5-(1H-imidazol-2-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-(1H-imidazol-4-yl)-2 -(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 5-(imidazo[1,2-a]pyrazin-3-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(5,6,7,8-tetrahydroimidazo[1,2-a]pyrazin-3-yl)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(4-methyl-1H-imidazol-2-yl)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1-methyl-1H-imidazol-4-yl)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(1-methyl-1H-imidazol-5-yl)phenol, 2-(6-(methyl(2 ,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(4-nitro-1H-imidazol-2-yl)phenol, 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-5-(2-methyl-1H-imidazol-4-yl)phenol, 5-(1,2-dimethyl-1H-imidazol-4-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 1-( 3-Hydroxy-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenyl)-1H-pyrazole-4-carboxamide, 2-(6-((3aR,6aS)-5-(2-hydroxyethyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridazin-3-yl)-5-(1H-pyrazol-4-yl)phenol, 2-(6-((3aR,6aS)-hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridazin-3-yl)-5- (1H-pyrazol-4-yl)phenol, 2-(6-((3aR,6aS)-5-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridazin-3-yl)-5-(1H-pyrazol-4-yl)phenol, 4-(3-hydroxy-4-(6-(5-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridazin-3-yl)phenyl)-1-methylpyridin-2(1H)-one, 4-(3-hydroxy-4-(6-((3aR,6aR)-1-methylhexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)pyridazin-3-yl)phenyl)-1-methylpyridin-2(1H)-one, 2-(6-(2,7-diazaspiro[4.5]decan-2-yl)pyridazin-3-yl)-5-(1H-pyrazol-4-yl)phenol, and 4-(4-(6-(2,7-diazaspiro[4.5]decan-2-yl)pyridazin-3-yl)-3-hydroxyphenyl)-1-methylpyridin-2(1H)-one.

[0132] Another representative splice modifier is RG7916 (Roche / PTC / SMAF), which has the following structure: 35 , 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one). TIFF2025060942000005.tif33137

[0133] Another representative splice modifying agent is RG7800 (Roche), which has the following structure: TIFF2025060942000006.tif48128

[0134] Also included are analogs of splice modifiers such as RG7916 and RG7800 that can be used similarly, such as, for example, the following: 2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-(4-methylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one, 7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 7-[(8aS)-8a-methyl-1,3,4,6,7,8-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one 7-[(8aR)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5R)-3,5-dimethylpiperazin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 7-(1,4-diazepan-1-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 7-(1,4-diazepan-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 7-[(3R,5S )-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 7-[(8aS)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1 ,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 7-[(8aR)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 7-(4,7-diazaspiro[2.5 ]octan-7-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one, 7-(3,3-dimethylpiperazin-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one, 7-(4,7- Diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a] Pyrimidin-4-one, 2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 7-(3,3-dimethylpiperazin-1-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido [1,2-a]pyrimidin-4-one, 7-(4,7-diazaspiro[2.5]octan-7-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, and 7-[(3R)-3-ethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one. 7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one Chilimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5R)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazine 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one, or a pharma- ceutically acceptable salt thereof.

[0135] Another representative family of splice modifiers are (5',Z)-5-(((lR,4R)-4-((2JE',4JE)-5-((3R,55')-7,7-dimethyl-l,6-dioxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dien-l-yl)cyclohexyl)amino)-5-oxopent-3-en-2-yl methylcarbamate and (5',Z)-5- The compound (sudemycin) disclosed in U.S. Pat. No. 9,682,993 includes (((lR,4R)-4-((2JE',4JE)-5-((3R,55')-7,7-dimethyl-l,6-dioxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dien-l-yl)cyclohexyl)amino)-5-oxopent-3-en-2-yl dimethylcarbamate.

[0136] Yet another representative family of splice modifying agents are the pladienolide compounds, which include those disclosed in the following patent applications: WO 2002 / 060890, WO 2004 / 011459, WO 2004 / 011661, WO 2004 / 050890, WO 2005 / 052152, WO 2006 / 009276, WO 2008 / 126918, and WO 2015 / 175594, each of which is incorporated herein by reference. One example of a pladienolide compound is (8E,12E,14E)-7-((4-cycloheptylpiperazin-1-yl)carbonyl)oxy-3,6,16,21-tetrahydroxy-6,10,12,16,20-pentamethyl-18,19-epoxytricosa-8,12,14-trien-11-olide, which is a semisynthetic derivative of the natural product pladienolide D and is also known as E7107.

[0137] III. Method of Administration Any suitable cell or mammal may be administered or treated by the methods or uses described herein. Typically, a mammal suspected of having or expressing an aberrant or abnormal protein associated with a disease state is in need of the methods described herein.

[0138] Non-limiting examples of mammals include humans, non-human primates (such as apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), livestock (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In certain embodiments, the mammal is a human. In certain embodiments, the mammal is a non-rodent mammal (e.g., a human, pig, goat, sheep, horse, dog, etc.). In certain embodiments, the non-rodent mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult, teenager, child, infant, or mammal in utero). The mammal can be male or female. In certain embodiments, the mammal can be an animal disease model, such as an animal model having or expressing an abnormal or aberrant protein associated with a disease state, or an animal model in which the expression of a protein is insufficient and causes the disease.

[0139] Mammals (subjects) treated with the methods or compositions described herein include adults (18 years and older) and children (under 18 years). Adults include the elderly. A typical adult is 50 years and older. Children range in age from 1-2 or 2-4 years, 4-6 years, 6-18 years, 8-10 years, 10-12 years, 12-15 years, and 15-18 years. Children also include infants. Infants are typically in the 1-12 month age range.

[0140] In certain embodiments, the method comprises administering a plurality of viral particles or nanoparticles to a mammal as described herein, and reducing, reducing, preventing, inhibiting, or delaying the severity, frequency, progression, or onset time of one or more symptoms of a disease state, such as a neurodegenerative disease.In certain embodiments, the method comprises administering a plurality of viral particles or nanoparticles to a mammal to treat a deleterious symptom of a disease state, such as a neurodegenerative disease.In certain embodiments, the method comprises administering a plurality of viral particles or nanoparticles to a mammal to stabilize, delay, or prevent the deterioration or progression or reversal and deleterious symptoms of a disease state, such as a neurodegenerative disease.

[0141] In certain embodiments, the methods include administering a plurality of viral particles or nanoparticles to the central nervous system or a portion thereof, as described herein, to reduce, reduce, prevent, inhibit, or delay by at least about 5 to about 10 days, about 10 to about 25 days, about 25 to about 50 days, or about 50 to about 100 days the severity, frequency, progression, or time to onset of one or more symptoms of a disease state, such as a neurodegenerative disease.

[0142] In certain embodiments, the symptoms or adverse effects include early symptoms, mid-stage or late stage symptoms, behavioral symptoms, personality or language symptoms, swallowing, movement, seizures, tremors or restlessness, ataxia, and / or cognitive symptoms such as memory and ability to organize.

[0143] In some embodiments, viral and non-viral gene transfer methods can be used to introduce nucleic acid into mammalian cells or mammalian target tissues.Such methods can be used to administer the nucleic acid that codes for inhibitory RNA, therapeutic protein, or CRISPR system components to cells in culture or in host organisms.Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcription product of vectors described herein), naked nucleic acid, and nucleic acid complexed with delivery vehicles such as liposomes.Viral vector delivery systems include DNA and RNA viruses that have episomal genomes or integrated genomes after delivery to cells. For reviews of gene therapy procedures, see Anderson, 1992, Nabel & Feigner, 1993, Mitani & Caskey, 1993, Dillon, 1993, Miller, 1992, Van Brunt, 1988, Vigne, 1995, Kremer & Perricaudet, 1995, Haddada et al., 1995, and Yu et al., 1994.

[0144] Methods for non-viral delivery of nucleic acid include exosomes, lipofection, nucleofection, microinjection, biolistic methods, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced DNA uptake.Lipofection has been described (e.g., U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355), and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™).Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91117424, WO 91116024.Delivery can be to cells (e.g., in vitro or ex vivo administration) or to target tissue (e.g., in vivo administration).

[0145] In some embodiments, delivery is by using RNA virus or DNA virus-based nucleic acid delivery system.In some aspects, viral vectors can be directly administered to patients (in vivo), or can be used to treat cells in vitro or ex vivo, and then administered to patients.In some embodiments, viral-based systems include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex viral vectors for gene transfer.

[0146] A. Viral Vectors The term "vector" refers to a small carrier nucleic acid molecule, a plasmid, a virus (e.g., AAV vector, retroviral vector, lentiviral vector), or other vehicle that can be manipulated by the insertion or incorporation of a nucleic acid. A vector, such as a viral vector, can be used to introduce / transfer a nucleic acid into a cell such that the nucleic acid sequence within the nucleic acid is transcribed by the cell and, if it encodes a protein, subsequently translated.

[0147] An "expression vector" is a special vector that contains a gene or nucleic acid sequence together with the necessary regulatory regions required for expression in a host cell. An expression vector may contain at least an origin of replication for propagation in a cell, and optionally additional elements, such as heterologous nucleic acid sequences, expression control elements (e.g., promoters, enhancers), introns, ITRs, and polyadenylation signals.

[0148] Viral vectors are derived from or based on one or more nucleic acid elements that make up the viral genome. Exemplary viral vectors include adeno-associated virus (AAV) vectors, retroviral vectors, and lentiviral vectors.

[0149] The term "recombinant" as a modifier of a vector, such as a recombinant viral vector, e.g., a recombinant lentivirus or recombinant parvovirus (e.g., AAV) vector, and as a modifier of a sequence, such as a recombinant nucleic acid sequence or a recombinant polypeptide, means that the composition has been manipulated (i.e., engineered) in a manner that does not generally occur in nature. A particular example of a recombinant vector, such as an AAV vector, a retrovirus vector, or a lentivirus vector, would be one in which a nucleic acid sequence not normally present in the wild-type viral genome has been inserted into the viral genome. An example of a recombinant nucleic acid sequence would be one in which a nucleic acid (e.g., a gene) encodes an inhibitory RNA cloned into the vector with or without the 5', 3', and / or intron regions that are normally associated with the gene in the viral genome. The term "recombinant" is not always used herein with respect to vectors, such as viral vectors, and sequences, such as polynucleotides, but "recombinant" forms, including nucleic acid sequences, polynucleotides, transgenes, etc., are clearly encompassed, even with such omissions.

[0150] A recombinant viral "vector" is derived from the wild-type genome of a virus, such as AAV, retrovirus or lentivirus, by using molecular methods to remove the wild-type genome from the virus and replace it with a non-native nucleic acid, such as a nucleic acid sequence. Typically, for example, in the case of AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. A "recombinant" viral vector (e.g., rAAV) is distinguished from a viral (e.g., AAV) genome in that all or part of the viral genome is replaced with a non-native sequence, such as a nucleic acid encoding a transactivator or a nucleic acid encoding an inhibitory RNA or a nucleic acid encoding a therapeutic protein, compared to the viral genome nucleic acid. Therefore, the incorporation of such a non-native nucleic acid sequence defines the viral vector as a "recombinant" vector, and in the case of AAV, it can be called a "rAAV vector".

[0151] 1. Adeno-associated virus Adeno-associated viruses (AAVs) are small, non-pathogenic viruses of the Parvoviridae family. To date, numerous serologically distinct AAVs have been identified, with more than a dozen identified from humans or primates. AAVs differ from other members of the family in that they depend on a helper virus for replication.

[0152] The AAV genome exists in an extrachromosomal state without integration into the host cell genome, has a broad host range, can transduce both dividing and non-dividing cells in vitro and in vivo, and can maintain high levels of expression of transduced genes. AAV viral particles are thermostable, resistant to solvents, detergents, changes in pH and temperature, and can be column purified and / or concentrated by CsCl gradients or other means. The AAV genome contains positive or negative single-stranded deoxyribonucleic acid (ssDNA). The approximately 5 kb genome of AAV consists of a segment of single-stranded DNA of positive or negative polarity. Both ends of the genome are short inverted terminal repeats (ITRs) that can fold into hairpin structures and serve as origins of viral DNA replication.

[0153] AAV "genome" refers to the recombinant nucleic acid sequence that is ultimately packaged or encapsidated to form an AAV particle. AAV particles often contain an AAV genome packaged with AAV capsid proteins. When a recombinant plasmid is used to construct or produce a recombinant vector, the AAV vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone," which is important for plasmid cloning and amplification, a process necessary for growth and recombinant virus production, but is not itself packaged or encapsidated into the viral particle. Thus, AAV vector "genome" refers to the nucleic acid that is packaged or encapsidated by the AAV capsid proteins.

[0154] AAV virions (particles) are non-enveloped icosahedral particles with a diameter of approximately 25 nm. AAV particles contain icosahedral symmetry composed of three related capsid proteins VP1, VP2, and VP3 that interact with each other to form the capsid. The right-hand ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins are often found in a ratio of 1:1:10, respectively, but the ratios can vary, and all originate from the right-hand ORF. The VP1, VP2, and VP3 capsid proteins differ from each other by alternative splicing and the use of unusual start codons. Deletion analysis has shown that removal or remodeling of VP1, which is translated from a message that undergoes alternative splicing, results in reduced yields of infectious particles. Mutations within the VP3 coding region result in single-stranded progeny DNA or failure to produce infectious particles.

[0155] An AAV particle is a viral particle that contains an AAV capsid. In certain embodiments, the genome of the AAV particle encodes one, two or all of the VP1, VP2 and VP3 polypeptides.

[0156] The genome of most native AAV often contains two open reading frames (ORFs), which are sometimes referred to as the left ORF and the right ORF. The left ORF often encodes the nonstructural Rep proteins Rep40, Rep52, Rep68 and Rep78, which are involved in regulating replication and transcription as well as producing single-stranded progeny genomes. Two of the Rep proteins have been associated with preferential inclusion of the AAV genome in a region of the q arm of human chromosome 19. Rep68 / 78 has been shown to have NTP binding activity as well as DNA and RNA helicase activity. Some Rep proteins have nuclear localization signals, along with several potential phosphorylation sites. In certain embodiments, the genome of an AAV (e.g., rAAV) encodes some or all of the Rep proteins. In certain embodiments, the genome of an AAV (e.g., rAAV) does not encode a Rep protein. In certain embodiments, one or more of the Rep proteins can be delivered in trans and are therefore not included in the AAV particle that contains the nucleic acid encoding the polypeptide.

[0157] The ends of the AAV genome contain short inverted terminal repeats (ITRs) that have the potential to fold into T-shaped hairpin structures that serve as origins for viral DNA replication. Thus, the genome of AAV contains one or more (e.g., a pair) ITR sequences that flank the single-stranded viral DNA genome. The ITR sequences are often approximately 145 bases long each. Within the ITR region, two elements that are thought to be central to ITR function have been described: the GAGC repeat motif and the terminal resolution site (trs). The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation. This binding is thought to position Rep68 / 78 for cleavage at the trs in a site- and strand-specific manner. In addition to their role in replication, these two elements appear to be central to viral inclusion. The chromosome 19 integration locus contains a Rep binding site flanked by trs. These elements have been shown to be functional and necessary for locus-specific inclusion.

[0158] In certain embodiments, an AAV (e.g., rAAV) comprises two ITRs. In certain embodiments, an AAV (e.g., rAAV) comprises a pair of ITRs. In certain embodiments, an AAV (e.g., rAAV) comprises a pair of ITRs adjacent to a nucleic acid sequence encoding a polypeptide having at least a function or activity (i.e., at the 5' and 3' ends of the nucleic acid sequence, respectively).

[0159] AAV vectors (e.g., rAAV vectors) can be packaged and are referred to herein as "AAV particles" for infecting (transducing) cells ex vivo, in vitro or in vivo. When a recombinant AAV vector is enclosed or packaged in an AAV particle, the particle can also be referred to as a "rAAV particle". In certain embodiments, the AAV particle is a rAAV particle. The rAAV particle often comprises a rAAV vector or a portion thereof. The rAAV particle can be one or more rAAV particles (e.g., multiple AAV particles). The rAAV particle typically comprises a protein (e.g., a capsid protein) that encloses or packages the rAAV vector genome. It should be noted that references to rAAV vectors can also be used to refer to rAAV particles.

[0160] Any suitable AAV particle (e.g., rAAV particle) can be used in the method or use herein. The rAAV particle and / or genome contained therein can be derived from any suitable serotype or strain of AAV. The rAAV particle and / or genome contained therein can be derived from more than one serotype or strain of AAV. Thus, the rAAV can comprise the protein and / or nucleic acid or a part thereof of any serotype or strain of AAV, and the AAV particle is suitable for infection and / or transduction of mammalian cells. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10 and AAV-2i8.

[0161] In certain embodiments, the plurality of rAAV particles comprises particles of the same strain or serotype (or subgroup or variant), or particles derived from the same strain or serotype (or subgroup or variant). In certain embodiments, the plurality of rAAV particles comprises a mixture of two or more different (e.g., different serotypes and / or different strains) rAAV particles.

[0162] As used herein, the term "serotype" is a characteristic used to refer to an AAV that has a capsid that is serologically different from other AAV serotypes. Serological characteristics are determined based on the lack of cross-reactivity between antibodies to one AAV compared to another AAV. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2 and / or VP3 sequences of AAV serotypes). Although AAV variants, including capsid variants, may not be serologically different from a reference AAV serotype or other AAV serotypes, they differ from the reference AAV serotype or other AAV serotypes by at least one nucleotide or amino acid residue.

[0163] In certain embodiments, rAAV particles exclude certain serotypes. In one embodiment, rAAV particles are not AAV4 particles. In certain embodiments, rAAV particles are antigenically or immunologically different from AAV4. Difference can be determined by standard methods. For example, ELISA and Western blot can be used to determine whether a viral particle is antigenically or immunologically different from AAV4. Furthermore, in certain embodiments, rAAV2 particles maintain a different tissue tropism from AAV4.

[0164] In certain embodiments, an rAAV vector based on a first serotype genome corresponds to one or more serotypes of the capsid proteins that package the vector. For example, the serotype of one or more AAV nucleic acids (e.g., ITRs) that make up the AAV vector genome corresponds to the serotype of the capsid that makes up the rAAV particle.

[0165] In certain embodiments, rAAV vector genome can be based on AAV (e.g., AAV2) serotype genome, which is derived from one or more serotypes of AAV capsid proteins that package the vector.For example, rAAV vector genome can include nucleic acid (e.g., ITR) from AAV2, while at least one or more of the three capsid proteins are derived from different serotypes, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 serotypes or their variants.

[0166] In certain embodiments, an rAAV particle or its vector genome associated with a reference serotype has a polynucleotide, polypeptide, or subsequence thereof that comprises or consists of a sequence that is at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a polynucleotide, polypeptide, or subsequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 particle. In certain embodiments, an rAAV particle or its vector genome related to a reference serotype has a capsid or ITR sequence that comprises or consists of a sequence that is at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to the capsid or ITR sequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 serotype.

[0167] In certain embodiments, the methods herein include use, administration or delivery of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74 or rAAV-2i8 particles.

[0168] In certain embodiments, the methods herein include the use, administration or delivery of rAAV2 particles.In certain embodiments, the rAAV2 particles include AAV2 capsid.In certain embodiments, the rAAV2 particles include one or more capsid proteins (e.g., VP1, VP2 and / or VP3) that are at least 60%, 65%, 70%, 75% or more identical to the corresponding capsid protein of native AAV2 particles or wild-type AAV2 particles, such as 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, and up to 100% identical. In certain embodiments, the rAAV2 particles comprise VP1, VP2 and VP3 capsid proteins that are at least 75% identical or greater to the corresponding capsid proteins of a native or wild-type AAV2 particle, e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical. In certain embodiments, the rAAV2 particles are variants of native or wild-type AAV2 particles. In some aspects, one or more capsid proteins of the AAV2 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to a capsid protein of a native or wild-type AAV2 particle.

[0169] In certain embodiments, the rAAV9 particles comprise an AAV9 capsid. In certain embodiments, the rAAV9 particles comprise one or more capsid proteins (e.g., VP1, VP2 and / or VP3) that are at least 60%, 65%, 70%, 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to the corresponding capsid protein of a native or wild-type AAV9 particle. In certain embodiments, the rAAV9 particles comprise VP1, VP2 and VP3 capsid proteins that are at least 75% identical or greater to the corresponding capsid proteins of a native or wild-type AAV9 particle, e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical. In certain embodiments, the rAAV9 particles are variants of native or wild-type AAV9 particles. In some aspects, one or more capsid proteins of the AAV9 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to a capsid protein of a native or wild-type AAV9 particle.

[0170] In certain embodiments, the rAAV particles are designed such that they possess one or more desired ITR functions (e.g., the ability to form a hairpin that allows DNA replication, the ability to integrate the AAV into the host cell genome, and the like). In some embodiments, the AAV-2i8 vector comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% identical to the corresponding ITRs of native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10 or AAV-2i8, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical, so long as the vector retains DNA inclusion, and / or packaging, if desired.

[0171] In certain embodiments, the rAAV2 particles contain one or two ITRs (e.g., a pair of ITRs) that are at least 75% identical or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical, to the corresponding ITRs of a native or wild-type AAV2 particle, so long as they retain one or more desired ITR functions (e.g., the ability to form a hairpin to enable DNA replication, inclusion of the AAV DNA into the host cell genome, and / or packaging if desired).

[0172] In certain embodiments, the rAAV9 particles contain one or two ITRs (e.g., a pair of ITRs) that are at least 75% identical or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical, to the corresponding ITRs of native or wild-type AAV2 particles, so long as they retain one or more desired ITR functions (e.g., the ability to form a hairpin to enable DNA replication, inclusion of the AAV DNA into the host cell genome, and / or packaging if desired).

[0173] rAAV particles can include an ITR with any suitable number of "GAGC" repeats. In certain embodiments, the ITR of an AAV2 particle includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more "GAGC" repeats. In certain embodiments, the rAAV2 particle includes an ITR with three "GAGC" repeats. In certain embodiments, the rAAV2 particle includes an ITR with fewer than four "GAGC" repeats. In certain embodiments, the rAAV2 particle includes an ITR with more than four "GAGC" repeats. In certain embodiments, the ITR of an rAAV2 particle includes a Rep binding site in which the fourth nucleotide in the first two "GAGC" repeats is C instead of T.

[0174] Examples of suitable lengths of DNA that can be incorporated into rAAV vectors for packaging / encapsidation into rAAV particles can be about 5 kilobases (kb) or less. In certain embodiments, the length of the DNA is less than about 5 kb, less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, or less than about 2.5 kb.

[0175] rAAV vectors containing nucleic acid sequences directing the expression of RNAi or polypeptides can be produced using suitable recombinant techniques known in the art (see, for example, Sambrook et al., 1989). Recombinant AAV vectors are typically packaged into transducible AAV particles and propagated using AAV viral packaging systems. Transducible AAV particles have the ability to bind and enter mammalian cells and subsequently deliver their nucleic acid cargo (e.g., heterologous genes) to the nucleus of the cells. Thus, intact transducible rAAV particles are configured to transduce mammalian cells. rAAV particles configured to transduce mammalian cells are often not replicable and require additional protein machinery to replicate themselves. Thus, rAAV particles configured to transduce mammalian cells are engineered to bind and enter mammalian cells and deliver nucleic acid to the cells, with the delivered nucleic acid often being located between a pair of AAV ITRs in the rAAV genome.

[0176] Suitable host cells for producing transducible AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can be used as or have been used as recipients of heterologous rAAV vectors. The stable human cell line HEK293 (e.g., readily available from the American Type Culture Collection under accession number ATCC CRL1573) can be used. In certain embodiments, modified human embryonic kidney cell lines (e.g., HEK293) that have been transformed with adenovirus type 5 DNA fragments and express adenovirus E1a and E1b genes are used to produce recombinant AAV particles. The modified HEK293 cell line is easily transfected, making it a particularly convenient platform for producing rAAV particles. Methods for producing high titer AAV particles capable of transducing mammalian cells are known in the art. For example, AAV particles can be made as described in Wright, 2008 and Wright, 2009.

[0177] In certain embodiments, AAV helper functions are introduced into host cells by transfecting AAV helper constructs into host cells before or simultaneously with transfection of AAV expression vector. Thus, in some cases, AAV helper constructs are used to at least transiently express AAV rep and / or cap genes to complement missing AAV functions required for productive AAV transduction. AAV helper constructs often lack AAV ITRs and cannot replicate or package themselves. These constructs can take the form of plasmids, phages, transposons, cosmids, viruses, or virions. Several AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which code for both Rep and Cap expression products. Several other vectors are also known that code for Rep and / or Cap expression products.

[0178] 2. Retroviruses In the methods, compositions and uses herein, viral vectors used as delivered agents include retroviral vectors (see, e.g., Miller (1992) Nature, 357:455-460). Retroviral vectors are well suited for delivering nucleic acids to cells because they have the ability to deliver unrearranged single copy genes to a wide range of rodent, primate and human somatic cells. Retroviral vectors are integrated into the genome of host cells. Unlike other viral vectors, they only infect dividing cells.

[0179] Retroviruses are RNA viruses, so their viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate that is integrated very efficiently into the chromosomal DNA of the infected cell. This integrated DNA intermediate is called a provirus. Transcription of the provirus and assembly into an infectious virus occurs in the presence of a suitable helper virus or in a cell line that contains the appropriate sequences that allow for packaging without the coproduction of a contaminating helper virus. If the sequences for packaging are provided by cotransfection of a suitable vector, a helper virus is not required for the production of recombinant retrovirus.

[0180] Retroviral genomes and proviral DNA have three genes, gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene codes for the internal structural (matrix, capsid and nucleocapsid) proteins, while the env gene codes for the viral envelope glycoprotein. The pol gene codes for products including the RNA-directed DNA polymerase reverse transcriptase, which transcribes the viral RNA into double-stranded DNA, integrase, which integrates the DNA produced by reverse transcriptase into the host chromosomal DNA, and protease, which acts to process the encoded gag and pol genes. The 5'LTR and 3'LTR serve to facilitate transcription and polyadenylation of virion RNA. The LTR contains all other cis-acting sequences necessary for viral replication.

[0181] Retroviral vectors are described in Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997). Representative examples of retroviruses are Moloney Murine Leukemia Virus (MMLV) or Murine Stem Cell Virus (MSCV). Retroviral vectors may be replication competent or replication defective. Typically, retroviral vectors are replication defective, in that the coding regions of genes required for a new round of virion replication and packaging have been deleted or replaced with other genes. As a result, the virus cannot continue the typical cell lysis pathway after infecting the initial target cell. Such retroviral vectors and the agents required to produce such viruses (e.g., packaging cell lines) are commercially available (see, e.g., retroviral vectors and retroviral systems, such as catalog numbers 634401, 631503, 631501, etc., available from Clontech (Clontech, Mountain View, Calif.)).

[0182] Such retroviral vectors can be produced as delivered agents by replacing the viral genes required for replication with the nucleic acid molecule to be delivered. The resulting genome contains LTRs at both ends with one or more desired genes between them. Methods for producing retroviruses are known to those of skill in the art (see, for example, published PCT application WO1995 / 026411). Retroviral vectors can be produced in packaging cell lines containing one or more helper plasmids. The packaging cell lines provide the viral proteins required for vector encapsidation and virion maturation (e.g., gag, pol and env genes). Typically, at least two separate helper plasmids (containing gag and pol genes separately from env genes) are used to ensure that recombination between vector plasmids cannot occur. For example, retroviral vectors can be transferred into packaging cell lines using standard transfection methods, such as calcium phosphate transfection. Packaging cell lines are well known to those of skill in the art and are commercially available. An exemplary packaging cell line is the GP2-293 packaging cell line (Catalog Nos. 631505, 631507, 631512, Clontech). After sufficient time for virion production, the virus is harvested. If desired, the harvested virus can be used to infect a second packaging cell line, for example to produce a virus with a different host tropism. The end result is a replicative incompetent recombinant retrovirus that contains the nucleic acid of interest but lacks other structural genes so that new viruses cannot form in the host cell.

[0183] References illustrating the use of retroviral vectors in gene therapy include Clowes et al., (1994) J. Clin. Invest. 93:644-651, Kiem et al., (1994) Blood 83:1467-1473, Salmons and Gunzberg (1993) Human Gene Therapy 4:129-141, Grossman and Wilson (1993) Curr. Opin. in Genetics and Devel. 3:110-114, Sheridan (2011) Nature Biotechnology, 29:121, Cassani et al. (2009) Blood, 114:3546-3556.

[0184] 3. Lentivirus Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. Their high complexity allows the virus to modulate its life cycle as seen in the course of latent infection. Examples of lentiviruses include human immunodeficiency viruses: HIV-1, HIV-2, and simian immunodeficiency viruses: SIV. Lentiviral vectors have been created by multiple attenuation of HIV virulence genes, for example by deleting genes env, vif, vpr, vpu, and nef, making the vector biologically safe. Lentiviral vectors are well known in the art (see, for example, U.S. Patent Nos. 6,013,516 and 5,994,136).

[0185] Recombinant lentivirus vectors have the ability to infect non-dividing cells and can be used for gene transfer and expression of nucleic acid sequences in vivo or ex vivo.For example, recombinant lentiviruses capable of infecting non-dividing cells, in which suitable host cells are transfected with two or more vectors carrying packaging functions, namely gag, pol and env, and rev and tat, are described in U.S. Patent No. 5,994,136, which is incorporated herein by reference.

[0186] Lentivirus genome and proviral DNA have three genes found in retroviruses, gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene codes for the internal structural (matrix, capsid and nucleocapsid) proteins, the pol gene codes for the RNA-directed DNA polymerase (reverse transcriptase), protease and integrase, and the env gene codes for the viral envelope glycoprotein. The 5'LTR and 3'LTR serve to facilitate transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx.

[0187] The 5'LTR is flanked by sequences required for reverse transcription of the genome (tRNA primer binding sites) and for efficient encapsidation of viral RNA into particles (Psi sites). When sequences required for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, a cis defect prevents encapsidation of genomic RNA; however, the resulting mutant still retains the ability to direct the synthesis of all virion proteins.

[0188] 4. Other viral vectors The development and utility of viral vectors for gene delivery is constantly improving and advancing. Other viral vectors, such as poxviruses, e.g., vaccinia virus (Gnant et al., 1999; Gnant et al., 1999), alphaviruses, e.g., Sindbis virus, Semliki Forest virus (Lundstrom, 1999), reoviruses (Coffey et al., 1998), and influenza A virus (Neumann et al., 1999), are also contemplated for use in the present disclosure and may be selected depending on the properties required for the targeting system.

[0189] 5. Chimeric viral vectors Chimeric or hybrid viral vectors have been developed for use in therapeutic gene delivery, and their use in the present disclosure is contemplated. Chimeric proviral / retroviral vectors (Holzer et al., 1999), adenoviral / retroviral vectors (Feng et al., 1997, Bilbao et al., 1997, Caplen et al., 2000) and adenoviral / adeno-associated viral vectors (Fisher et al., 1996, U.S. Patent No. 5,871,982) have been described. These "chimeric" viral gene transfer systems can take advantage of the favorable characteristics of two or more parental viral species. For example, Wilson et al. provide the following chimeric vector constructs, which include a portion of adenovirus, 5'ITR and 3'ITR sequences of AAV, and a selected transgene (U.S. Patent No. 5,871,983, which is specifically incorporated herein by reference).

[0190] B. Nanoparticles 1. Lipid-based nanoparticles In some embodiments, the lipid-based nanoparticle is a liposome, an exosome, a lipid preparation, or other lipid-based nanoparticle, such as a lipid-based vesicle (e.g., DOTAP:cholesterol vesicle). The lipid-based nanoparticle can be positively charged, negatively charged, or neutral.

[0191] Liposomes "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid media formed by the formation of closed lipid bilayers or lipid aggregates. Liposomes can be characterized as having a vesicular structure with a bilayer membrane that generally comprises phospholipids and an inner medium that generally comprises an aqueous composition. Liposomes provided herein include unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. Liposomes provided herein can be positively, negatively, or neutrally charged. In certain embodiments, liposomes are neutrally charged.

[0192] Multilamellar liposomes have multiple lipid layers separated by aqueous media. Such liposomes form spontaneously when lipids, including phospholipids, are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures, trapping water and dissolved solutes between the lipid bilayers. Lipophilic molecules or molecules with lipophilic regions can also dissolve in or associate with the lipid bilayer.

[0193] In specific aspects, the polypeptide, nucleic acid or small molecule drug can be, for example, encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule that is associated with both the liposome and the polypeptide / nucleic acid, entrapped within the liposome, or complexed with the liposome.

[0194] Liposomes used according to this embodiment can be made in a variety of ways that will be known to those skilled in the art. For example, phospholipids, such as the neutral phospholipid dioleoylphosphatidylcholine (DOPC), are dissolved in tert-butanol. The lipids are then mixed with polypeptides, nucleic acids and / or other components. Tween 20 is added to the lipid mixture such that Tween 20 is about 5% of the composition weight. An excess of tert-butanol is added to the mixture such that the volume of tert-butanol is at least 95%. The mixture is vortexed, frozen in a dry ice / acetone bath, and lyophilized overnight. The lyophilized preparations are stored at -20°C and can be used for up to 3 months. When necessary, the lyophilized liposomes are reconstituted in 0.9% saline.

[0195] Alternatively, liposomes can be prepared by mixing lipids in a solvent in a vessel such as a glass pear-shaped flask. The vessel should have a volume ten times larger than the volume of the expected liposome suspension. The solvent is removed using a rotary evaporator under reduced pressure at approximately 40°C. The solvent is usually removed within about 5 minutes to 2 hours, depending on the volume of liposomes desired. The composition can be further dried under vacuum in a desiccator. Dried lipids tend to deteriorate over time, so are generally discarded after about one week.

[0196] The dried lipids can be hydrated in sterile pyrogen-free water at approximately 25-50 mM phospholipid by shaking until the lipid film is resuspended. The aqueous liposomes can then be aliquoted and placed into vials, lyophilized, and sealed under vacuum.

[0197] The dried lipids or lyophilized liposomes prepared as described above are dehydrated and may be reconstituted in a solution of protein or peptide and diluted to an appropriate concentration with a suitable solvent, such as DPBS. The mixture is then vigorously stirred on a vortex mixer. Any unencapsulated additional materials, such as agents including but not limited to hormones, drugs, nucleic acid constructs, etc., are removed by centrifugation at 29,000×g, and the liposome pellet is washed. The washed liposomes are resuspended at an appropriate total lipid concentration, such as about 50-200 mM. The amount of encapsulated additional materials or additional active agents can be determined according to standard methods. After determining the amount of additional materials or additional active agents encapsulated in the liposome preparation, the liposomes can be diluted to an appropriate concentration and stored at 4° C. until use. Pharmaceutical compositions containing liposomes will usually include a sterile pharma- ceutical acceptable carrier or diluent, such as water or saline solution.

[0198] Additional liposomes that may be useful in this embodiment include cationic liposomes, such as those described in WO02 / 100435A1, U.S. Pat. No. 5,962,016, U.S. Patent Application Publication No. 2004 / 0208921, WO03 / 015757A1, WO04029213A2, U.S. Pat. No. 5,030,453, and U.S. Pat. No. 6,680,068, which are incorporated by reference in their entireties without exception.

[0199] When preparing such liposomes, any protocol described herein or any protocol that would be known to those skilled in the art can be used.Further non-limiting examples of liposome preparation are described in U.S. Patent Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505 and 4,921,706, and International Application PCT / US85 / 01161 and PCT / US89 / 05040, each of which is incorporated herein by reference.

[0200] In certain embodiments, the lipid-based nanoparticle is a neutral liposome (e.g., DOPC liposome). As used herein, "neutral liposome" or "uncharged liposome" is defined as a liposome that has one or more lipid components that provide an essentially neutral net charge (substantially uncharged). "Essentially neutral" or "essentially uncharged" means that few, if any, lipid components in a given population (e.g., a population of liposomes) contain a charge that is not counterbalanced by the opposite charge of another component (i.e., less than 10% of the components, more preferably less than 5%, and most preferably less than 1% contain a charge that is not counterbalanced). In certain embodiments, neutral liposomes may primarily comprise lipids and / or phospholipids that are themselves neutral at physiological conditions (i.e., about pH 7).

[0201] The liposomes and / or lipid-based nanoparticles of this embodiment may include phospholipids. In certain embodiments, one type of phospholipid may be used to create the liposomes (e.g., neutral phospholipids such as DOPC may be used to create neutral liposomes). In other embodiments, two or more types of phospholipids may be used to create the liposomes. The phospholipids may be derived from natural or synthetic sources. Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine. Phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., about pH 7), so these compounds may be particularly useful for creating neutral liposomes. In certain embodiments, the phospholipid DOPC is used to produce uncharged liposomes. In certain embodiments, lipids that are not phospholipids (e.g., cholesterol) may be used.

[0202] Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilaurylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoyl-2-myri ... 1-Palmitoyl-2-stearoylphosphatidylcholine ("PMPC"), 1-Palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-Stearoyl-2-Palmitoylphosphatidylcholine ("SPPC"), Dilauryl oil phosphatidylglycerol ("DLPG"), Dimyristoyl phosphatidylglycerol ("DMPG"), Dipalmitoyl phosphatidylglycerol ("DPPG"), Distearoyl phosphatidylglycerol ("DSPG"), PG"), distearoyl sphingomyelin ("DSSP"), distearoylphosphatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), dimyris ... Sphatidylserine ("DMPS"), dipalmitoylphosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoyl sphingomyelin ("DPSP"), dimyristylphosphatidylcholine ("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), 1,These include, but are not limited to, 2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloleoylphosphatidylcholine ("POPC"), palmitoyloleoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

[0203] b. Exosomes "Extracellular vesicles" and "EVs" refer to cell-derived and cell-secreted microvesicles, which include types such as exosomes, exosome-like vesicles, ectosomes (which arise by vesicle budding directly from the plasma membrane), microparticles, microvesicles, shedding microvesicles (SMVs), nanoparticles, as well as (large) apoptotic blebs or bodies (which arise from cell death) or membrane particles.

[0204] As used herein, the terms "microvesicle" and "exosome" refer to a membrane particle having a diameter (or maximum dimension if the particle is not a spheroid) of about 10 nm to about 5000 nm, more typically 30 nm to 1000 nm, and most typically about 50 nm to 750 nm, where at least a portion of the membrane of the exosome is obtained directly from a cell. Most commonly, the exosome will have a size (average diameter) of up to 5% of the size of the donor cell. Thus, exosomes of particular interest include those shed from cells.

[0205] Exosomes may be detected in or isolated from any suitable sample type, such as, for example, bodily fluids. As used herein, the term "isolated" refers to separation from its natural environment, and includes at least partial purification, and may include substantial purification. As used herein, the term "sample" refers to any sample suitable for the methods provided by the present invention. The sample may be any sample that contains exosomes suitable for detection or isolation. Sources of samples include blood, bone marrow, pleural fluid, peritoneal fluid, cerebrospinal fluid, urine, saliva, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, milk, sweat, tears, joint fluid, and bronchial lavage fluid. In one aspect, the sample is a blood sample, including, for example, whole blood or any fraction or component thereof. Blood samples suitable for use in the present invention may be extracted from any known source that contains blood cells or components thereof, such as, for example, venous blood, arterial blood, peripheral blood, tissue blood, umbilical cord blood, etc. For example, samples can be collected and processed using well-known routine clinical methods (e.g., procedures for collecting and processing whole blood). In one aspect, an exemplary sample can be peripheral blood drawn from a subject with cancer.

[0206] Exosomes may also be isolated from tissue samples, such as surgical samples, biopsy samples, tissues, feces, and cultured cells. When isolating exosomes from tissue sources, it may be necessary to homogenize the tissue to obtain a single cell suspension and then lyse the cells to release the exosomes. When isolating exosomes from tissue samples, it is important to select homogenization and lysis techniques that do not result in exosome fragmentation. The exosomes contemplated herein are preferably isolated from body fluids into physiologically acceptable solutions, such as buffered saline, growth media, various aqueous media, and the like.

[0207] Exosomes may be isolated from freshly collected samples or from samples that have been frozen or refrigerated. In some embodiments, exosomes may be isolated from cell culture media. Although not necessary, more pure exosomes may be obtained if the liquid sample is clarified prior to precipitation with a volume-excluding polymer to remove any debris from the sample. Clarification methods include centrifugation, ultracentrifugation, filtration, or ultrafiltration. Most typically, exosomes can be isolated by a number of methods well known in the art. One preferred method is differential centrifugation from body fluids or cell culture supernatants. Exemplary exosome isolation methods are described in (Losche et al., 2004; Mesri and Altieri, 1998; Morel et al., 2004). Alternatively, exosomes may be isolated by flow cytometry, as described in (Combes et al., 1997).

[0208] One commonly accepted protocol for exosome isolation involves ultracentrifugation, which is often combined with a sucrose density gradient or sucrose cushion to float the relatively low density exosomes. Isolation of exosomes by differential sequential centrifugation is made difficult by the potential overlap of size distribution with other microvesicles or macromolecular complexes. Furthermore, centrifugation may be insufficient to separate vesicles based on their size. However, sequential centrifugation can be combined with sucrose gradient ultracentrifugation to obtain a high enrichment of exosomes.

[0209] Size-based exosome isolation using alternative methods to ultracentrifugation is another option. Successful purification of exosomes using ultrafiltration techniques, which require less time than ultracentrifugation and do not require specialized equipment, has been reported. Similarly, commercial kits are available that use positive pressure to drive fluids, allowing the removal of cells, platelets, and cell debris in one microfilter and the capture of vesicles larger than 30 nm in a second microfilter (EXOMIR™, Bioo Scientific). However, in this process, exosomes are not recovered, and their RNA content is directly extracted from the material captured on the second microfilter, which can be used for PCR analysis. HPLC-based protocols could potentially allow for highly pure exosomes, but these processes require dedicated equipment and are difficult to scale up. A significant problem is that both blood and cell culture media contain a large number of nanoparticles, some of which are non-vesicular, in the same size range as exosomes. For example, some miRNAs may be contained within extracellular protein complexes other than exosomes, but treatment with proteases (e.g., proteinase K) can remove possible "exosomal" protein contamination.

[0210] In another embodiment, exosomes may be captured by techniques commonly used to enrich samples for exosomes, such as techniques involving immunospecific interactions (e.g., immunomagnetic capture). Immunomagnetic capture, also known as immunomagnetic cell separation, typically involves attaching antibodies to proteins found on specific cell types to small paramagnetic beads. When the antibody-coated beads are mixed with a sample, such as blood, they bind to and surround the specific cells. The sample is then placed in a strong magnetic field, which causes the beads to pellet in one direction. The captured cells are retained with the beads after removal of the blood. Many variations of this general method are known in the art and are suitable for use in isolating exosomes. In one example, exosomes are bound to magnetic beads (e.g., aldehyde / sulfate beads) and then antibodies are added to the mixture to recognize epitopes on the surface of the exosomes bound to the beads.

[0211] As will be appreciated by those skilled in the art, exosomes may be further modified by including targeting moieties before or after loading with cargo to enhance their usefulness as vehicles for delivering cargo. In this regard, exosomes may be engineered to incorporate entities that specifically target certain cell or tissue types. This target-specific entity, e.g., a peptide with affinity for a receptor or ligand on a target cell or tissue, may be incorporated into the membrane of the exosome, e.g., by fusion to an exosome membrane marker using methods established in the art.

[0212] 2. Non-lipid nanoparticles Spherical Nucleic Acid (SNA™) constructs and other nanoparticles (especially gold nanoparticles) are also considered as a means to deliver chimeric minigenes to intended target cells. Because of their tight packing, the majority of the cargo (e.g., DNA) remains bound to the construct inside the cell, conferring stability and resistance to enzymatic degradation to the nucleic acid. In all cell types studied (e.g., neurons, tumor cell lines, etc.), these constructs show 99% transfection efficiency without the need for a single or transfection agent. The unique target binding affinity and specificity of the constructs allows for excellent specificity (i.e., limited off-target effects) for the matched target sequence. These constructs are significantly superior to the leading conventional transfection reagents (Lipofectamine 2000 and Cytofectin). These constructs can enter a variety of cultured cells, primary cells, and tissues without obvious toxicity. These constructs induce only modest changes in global gene expression as measured by whole genome microarray studies and cytokine-specific protein assays. Any number of single or combination agents (e.g., proteins, peptides, small molecules) can be used to tailor the surface of these constructs. See, e.g., Jensen et al., Sci. Transl. Med. 5, 209ra152 (2013).

[0213] Self-assembling nanoparticles carrying nucleic acid cargo can be constructed using PEGylated polyethyleneimine (PEI) with Arg-Gly-Asp (RGD) peptide ligands attached to the distal end of polyethylene glycol (PEG). Nanoplexes can be prepared by mixing equal volumes of aqueous solutions of cationic polymer and nucleic acid, with a net molar excess of ionizable nitrogen (polymer) over phosphate (nucleic acid) ranging from 2 to 6. Electrostatic interactions between the cationic polymer and nucleic acid led to the formation of polyplexes, which have a mean particle size distribution of about 100 nm and are therefore referred to as nanoplexes (see, for example, Bartlett et al., PNAS, 104:39, 2007).

[0214] C. Encapsulated Cell Transplantation The chimeric minigene herein can be delivered to cells ex vivo, which are then encapsulated and implanted to deliver the target gene to a patient. For example, exogenous heterologous nucleic acid can be introduced into cells isolated from a patient or donor and delivered directly to a patient by implanting the encapsulated cells. The advantage of implanting encapsulated cells is that immune responses against the cells are reduced by encapsulation. Thus, a method of symmetrically administering genetically modified cells is provided herein. The number of cells delivered depends on the desired effect, the specific nucleic acid, the subject to be treated, and other similar factors, and can be determined by one skilled in the art.

[0215] The cells into which nucleic acid is introduced for gene therapy include any desired available cell type, including, but not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells, such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, particularly hematopoietic stem cells or hematopoietic progenitor cells, such as those obtained from bone marrow, umbilical cord blood, peripheral blood, or fetal liver. For example, the genetically modified cells can be pluripotent or totipotent stem cells (including induced pluripotent stem cells), embryonic cells, fetal cells, or fully differentiated cells. The genetically modified cells can be cells from the same subject as the recipient subject, or cells from the same or different species as the recipient subject. In a preferred example, the cells used for gene therapy are autologous cells for the patient. Methods for genetically modifying cells and transplanting cells are known in the art.

[0216] Typically, the nucleic acid is first introduced into a cell, and then the resulting recombinant cell is administered in vivo. Such introduction can be carried out by any method known in the art, including but not limited to, transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques for introducing foreign genes into cells are known in the art (see, for example, Loeffler and Behr, Meth. Enzymol. (1993) 217:599-618; Cotten et al., Meth. Enzymol. (1993) 217:618-644; Cline, Pharmac. Ther. (1985) 29:69-92), and can be used as long as they do not disrupt the necessary developmental and physiological functions of the recipient cell. In certain instances, the methods allow for the stable transfer of a nucleic acid into a cell, such that the nucleic acid is expressible by the cell and capable of being inherited and expressed by its progeny cells.

[0217] Encapsulation can be achieved using alginate microcapsules coated with alginate / polylysine complexes. Hydrogel microcapsules have been widely studied for encapsulation of living cells or cell aggregates for tissue engineering and regenerative medicine (Orive, et al. Nat. Medicine 2003, 9, 104; Paul, et al., Regen. Med. 2009, 4, 733; Read, et al. Biotechnol. 2001, 19, 29). In general, capsules are designed to allow easy diffusion of oxygen and nutrients to the encapsulated cells while releasing therapeutic proteins secreted by the cells, and to protect the cells from attack by the immune system. They have been developed as potential therapeutics for a range of diseases including type I diabetes, cancer and neurodegenerative disorders such as Parkinson's disease (Wilson et al. Adv. Drug. Deliv. Rev. 2008, 60, 124; Joki, et al. Nat. Biotech. 2001, 19, 35; Kishima, et al. Neurobiol. Dis. 2004, 16, 428). One of the most common capsule formulations is based on alginate hydrogels, which can be formed by ionic crosslinking. In a typical process, cells are first combined with a viscous alginate solution. The cell suspension is then processed into microdroplets using a variety of methods, including air shear, acoustic vibration or electrostatic droplet formation (Rabanel et al. Biotechnol. Prog. 2009, 25, 946). The alginate droplets gel when contacted with a solution of divalent ions such as Ca2+ or Ba2+.

[0218] A capsule for implanting mammalian cells in a subject is disclosed. The capsule is formed from a biocompatible hydrogel-forming polymer that encapsulates the implanted cells. To inhibit capsular overgrowth (fibrosis), the capsule structure prevents cellular material from locating on the capsule surface. In addition, the capsule structure ensures that sufficient gas exchange occurs with the cells and that the cells encapsulated therein receive nutrients. Optionally, the capsule also contains one or more anti-inflammatory drugs entrapped therein for sustained release.

[0219] The disclosed composition is formed from a biocompatible hydrogel-forming polymer that encapsulates the cells to be implanted. Examples of materials that can be used to form suitable hydrogels include polysaccharides such as alginate, collagen, chitosan, sodium cellulose sulfate, gelatin and agarose, water-soluble polyacrylates, polyphosphatides, poly(acrylic acid), poly(methacrylic acid), poly(alkylene oxide), poly(vinyl acetate), polyvinylpyrrolidone (PVP), and copolymers and blends thereof. See, for example, U.S. Patent Nos. 5,709,854, 6,129,761, 6,858,229 and 9,555,007.

[0220] IV. Pharmaceutical Compositions As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to a biologically acceptable composition, formulation, liquid or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or in vivo contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects. Such compositions, "pharmaceutically acceptable" and "physiologically acceptable" formulations and compositions may be sterile. Such pharmaceutical formulations and pharmaceutical compositions may be used, for example, in administering viral particles or nanoparticles to a subject.

[0221] Such formulations and compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspending media, coatings, isotonicity and absorption enhancing or absorption delaying agents that are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions can include suspending agents and thickening agents. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the formulations and compositions.

[0222] Pharmaceutical compositions typically contain pharma- ceutically acceptable excipients. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition and that may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as sorbitol, Tween 80, water, saline, glycerol, and ethanol. They may include pharma-ceutically acceptable salts, such as mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, and salts of organic acids, such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as surfactants, wetting or emulsifying agents, pH buffering substances, and the like, may also be present in such vehicles.

[0223] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as indicated herein or known to those of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration or delivery by various routes.

[0224] Pharmaceutical dosage forms suitable for injection of viral particles or nanoparticles can include sterile aqueous solutions or dispersions adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In any case, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture, use, and storage. Liquid carriers or vehicles can be solvents or liquid dispersion media, including, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Isotonicity agents can be included, such as sugars, buffers, or salts (such as sodium chloride). Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0225] Solutions or suspensions of viral particles or nanoparticles may optionally contain one or more of the following components: sterile diluents, such as water for injection, saline solutions, such as phosphate-buffered saline (PBS), artificial CSF, surfactants, fixed oils, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol), glycerin, or other synthetic solvents, antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and the like; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose.

[0226] Pharmaceutical formulations, compositions and delivery systems suitable for the compositions, methods and uses of the invention are known in the art (e.g., Remington: The Science and Practice of Pharmacy (2003) 20). th ed., Mack Publishing Co., Easton, PA, Remington's Pharmaceutical Sciences (1990) 18 th ed., Mack Publishing Co., Easton, PA, The Merck Index (1996) 12 th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, PA; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11 thed., Lippincott Williams & Wilkins, Baltimore, Md., and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, New York, pp. 253-315).

[0227] The viral particles, nanoparticles and compositions thereof can be formulated into dosage unit forms for ease of administration and uniformity of dosage. As used herein, "dosage unit forms" refer to physically discrete units suitable as unitary dosages to treated individuals, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect together with the necessary pharmaceutical carrier. The dosage unit form depends on the number of viral particles or nanoparticles that are considered necessary to produce a desired effect. The required amount can be formulated in a single dose or in multiple dosage units. The dose can be adjusted to provide the appropriate viral particle or nanoparticle concentration, optionally combined with an anti-inflammatory agent, and packaged for use.

[0228] In one embodiment, the pharmaceutical composition will contain sufficient genetic material to provide a therapeutically effective amount, i.e., an amount sufficient to reduce or ameliorate the symptoms or deleterious effects of the disease state in question, or an amount sufficient to provide the desired benefit.

[0229] As used herein, "unit dosage form" refers to a physically discrete unit suitable for a unitary dosage to a subject to be treated, each unit containing a predetermined amount calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect) when administered once or multiple times, optionally together with a pharmaceutical carrier (excipient, diluent, vehicle or filler). Unit dosage forms may be, for example, in ampoules and vials that may contain liquid compositions, compositions in a freeze-dried or lyophilized state, and a sterile liquid carrier, for example, may be added before in vivo administration or in vivo delivery. Individual unit dosage forms may be included in a multi-dose kit or container. Thus, for example, viral particles, nanoparticles and pharmaceutical compositions thereof may be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0230] A formulation containing viral particles or nanoparticles typically contains an effective amount, which is easily determined by one skilled in the art. The viral particles or nanoparticles can typically be in the range of about 1% to about 95% (w / w) of the composition, or even higher if appropriate. The amount administered depends on factors such as the age, weight and health of the mammalian or human subject for which treatment is being considered. One skilled in the art can establish effective dosages by routine testing to establish dose-response curves.

[0231] V. Definition The terms "polynucleotide", "nucleic acid" and "transgene" are used interchangeably herein to refer to any form of nucleic acid, oligonucleotide, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and polymers thereof. Polynucleotides include genomic DNA, cDNA and antisense DNA, as well as spliced ​​or unspliced ​​mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-spliced ​​RNA or antisense RNA). Polynucleotides can include natural, synthetic and intentionally modified or altered polynucleotides (e.g., variant nucleic acids). Polynucleotides can be single-stranded, double-stranded or triplexed, linear or circular, and of any suitable length. In discussing polynucleotides, the sequence or structure of a particular polynucleotide may be described herein according to the convention of describing the sequence in the 5'→3' direction.

[0232] A nucleic acid that encodes a polypeptide often includes an open reading frame that encodes the polypeptide. Unless otherwise indicated, a particular nucleic acid sequence also includes degenerate codon substitutions.

[0233] The nucleic acid can include one or more expression control or expression regulatory elements operably linked to the open reading frame, which one or more regulatory elements are configured to direct the transcription and translation of the polypeptide encoded by the open reading frame in a mammalian cell. Non-limiting examples of expression control / regulatory elements include transcription initiation sequences (e.g., promoters, enhancers, TATA boxes, etc.), translation initiation sequences, mRNA stability sequences, polyA sequences, secretion sequences, etc. Expression control / regulatory sequences can be obtained from the genome of any suitable organism.

[0234] "Promoter" refers to a nucleotide sequence, usually upstream (5') of a coding sequence, that directs and / or controls the expression of the coding sequence by providing a recognition site for RNA polymerase and other factors necessary for proper transcription. "Promoter" includes minimal promoters, which are short DNA sequences consisting of a TATA box and optionally other sequences that serve to specify the transcription start site, to which regulatory elements are added to control expression.

[0235] An "enhancer" is a DNA sequence capable of stimulating transcriptional activity and can be a promoter-specific or heterologous element that enhances the level or tissue specificity of expression. It can operate in either orientation (5'→3' or 3'→5') and has the ability to function whether placed upstream or downstream of the promoter.

[0236] Promoters and / or enhancers may be derived in their entirety from a native gene, may be composed of different elements derived from different elements found in nature, or may even be composed of synthetic DNA segments. A promoter or enhancer may contain DNA sequences involved in the binding of protein factors that regulate / control the effectiveness of transcription initiation in response to stimuli, physiological or developmental conditions.

[0237] Non-limiting examples include SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, pol II promoter, pol III promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences from non-viral genes, such as the mouse metallothionein gene, will also be useful herein. Exemplary constitutive promoters include promoters of the following genes that code for certain constitutive or "housekeeping" functions, as well as other constitutive promoters known to those skilled in the art: hypoxanthine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, actin promoter. In addition, many viral promoters function constitutively in eukaryotic cells. These include, among others, the early and late promoters of SV40, the long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus, etc. Thus, any of the above-mentioned constitutive promoters can be used to control the transcription of the heterologous gene insert.

[0238] For convenience, the term "transgene" is used herein to refer to a nucleic acid sequence / polynucleotide that is or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as an inhibitory RNA or a gene encoding a polypeptide or protein, which is generally heterologous to the native AAV genome sequence.

[0239] The term "transduction" refers to the introduction of a nucleic acid sequence into a cell or host organism by a vector (e.g., a viral particle). Thus, the introduction of a transgene into a cell by a viral particle can be referred to as "transduction" of the cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. If the introduced transgene is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in that cell or organism and can be further transmitted or inherited by the descendant cells or organisms of the recipient cell or organism. Finally, the introduced transgene may be present extrachromosomally or only transiently in the recipient cell or recipient host organism. Thus, a "transduced cell" is a cell into which a transgene has been introduced by transduction. Thus, a "transduced" cell is a cell into which a transgene has been introduced or a descendant thereof. The transduced cell can be propagated, transcribed, and express the encoded inhibitory RNA or protein. For gene therapy uses and methods, the transduced cell can be present in a mammal.

[0240] A transgene under the control of an inducible promoter is expressed only in the presence of an inducer or is expressed more strongly in the presence of the inducer (e.g., transcription under the control of the metallothionein promoter is significantly increased in the presence of certain metal ions). Inducible promoters contain a responsive element (RE) that stimulates transcription when the respective inducer binds. Examples include REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. Promoters containing a specific RE can be chosen to obtain an inducible response, and in some cases, the RE itself can be attached to a different promoter to confer inducibility to the recombinant gene. Thus, by selecting the appropriate promoter (constitutive or inducible, strong or weak promoter), it is possible to control both the presence and the expression level of a polypeptide in a genetically modified cell. When a gene encoding a polypeptide is under the control of an inducible promoter, delivery of the polypeptide in situ is triggered by exposing the genetically modified cell in situ to conditions that allow transcription of the polypeptide, for example, by intraperitoneal injection of a specific inducer of the inducible promoter that controls the transcription of the agent. For example, in situ expression by genetically modified cells of a polypeptide encoded by a gene under the control of a metallothionein promoter is enhanced by contacting the genetically modified cells in situ with a solution containing the appropriate (i.e., inducible) metal ion.

[0241] A nucleic acid / transgene is said to be "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. The nucleic acid / transgene encoding an RNAi or polypeptide or the nucleic acid directing the expression of a polypeptide may contain an inducible promoter or a tissue-specific promoter to control the transcription of the encoded polypeptide. A nucleic acid operably linked to an expression control element may also be referred to as an expression cassette.

[0242] In certain embodiments, the methods and uses described herein use CNS-specific or inducible promoters, enhancers, etc. Non-limiting examples of CNS-specific promoters include those isolated from the genes of myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE).Non-limiting examples of inducible promoters include DNA responsive elements to ecdysone, tetracycline, hypoxia and IFN.

[0243] In certain embodiments, the expression control element comprises a CMV enhancer. In certain embodiments, the expression control element comprises a β-actin promoter. In certain embodiments, the expression control element comprises a chicken β-actin promoter. In certain embodiments, the expression control element comprises a CMV enhancer and a chicken β-actin promoter.

[0244] As used herein, the term "modify" or "variant" and its grammatical variants refer to nucleic acid, polypeptide or its subsequence deviating from reference sequence.Therefore, modified and variant sequences may have substantially the same, greater or less expression, activity or function compared to reference sequence, but at least partially retain the activity or function of reference sequence.A particular type of variant is mutant protein, which refers to the protein encoded by gene with mutation, such as missense mutation or nonsense mutation.

[0245] A "nucleic acid" variant or a "polynucleotide" variant refers to a modified sequence that is genetically altered compared to the wild type. A sequence can be genetically altered without altering the encoded protein sequence. Alternatively, a sequence can be genetically altered to encode a variant protein. A nucleic acid variant or a polynucleotide variant can also refer to a combination sequence that is codon-altered to encode a protein that still retains at least partial sequence identity to a reference sequence, such as a wild type protein sequence, and is also codon-altered to encode a variant protein. For example, some codons of such a nucleic acid variant will be altered so as not to alter the amino acid of the protein encoded thereby, and some codons of a nucleic acid variant will be altered so as to alter the amino acid of the protein that it encodes.

[0246] The terms "protein" and "polypeptide" are used interchangeably herein. The "polypeptide" encoded by the "nucleic acid" or "polynucleotide" or "transgene" disclosed herein includes partial or full-length native sequences, as well as naturally occurring wild-type and functional polymorphic proteins, their functional subsequences (fragments), and sequence variants thereof, so long as the polypeptide retains some function or activity. Thus, in the methods and uses of the present invention, such polypeptides encoded by nucleic acid sequences need not be identical to endogenous proteins that are defective or whose activity, function or expression is insufficient, deficient, or absent in the mammal being treated.

[0247] Non-limiting examples of modifications include substitution of one or more nucleotides or amino acids (e.g., about 1 to about 3, about 3 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 500, about 500 to about 750, about 750 to about 1000 or more nucleotides or residues).

[0248] Examples of amino acid modifications are conservative amino acid substitutions or deletions. In certain embodiments, the modified or variant sequence retains at least part of the function or activity of the unmodified sequence (e.g., the wild-type sequence).

[0249] Another example of amino acid modification is the targeting peptide that is introduced into the capsid protein of viral particles.Identified peptides target recombinant viral vectors or nanoparticles to the central nervous system, for example, vascular endothelial cells.Therefore, modified recombinant viral particles or nanoparticles can target, for example, the endothelial cells that line the blood vessels of the brain.

[0250] Such modified recombinant viruses may preferentially bind to one type of tissue (e.g., CNS tissue) over another type of tissue (e.g., liver tissue). In certain embodiments, recombinant viruses bearing modified capsid proteins may "target" cerebrovascular epithelial tissue by binding at a higher level than comparable unmodified capsid proteins. For example, recombinant viruses with modified capsid proteins may bind to cerebrovascular epithelial tissue at a 50%-100% higher level than unmodified recombinant viruses.

[0251] A "nucleic acid fragment" is a portion of a given nucleic acid molecule. In most organisms, deoxyribonucleic acid (DNA) is the genetic material, while ribonucleic acid (RNA) is responsible for the transfer of information contained in DNA into proteins. Fragments and variants of the disclosed nucleotide sequences and the proteins or partial length proteins encoded thereby are also encompassed by the present invention. By "fragment" or "portion" is meant a full-length or less than full-length nucleotide sequence encoding a polypeptide or protein, or an amino acid sequence of a polypeptide or protein. In certain embodiments, the fragment or portion is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the wild-type activity or function).

[0252] A "variant" of a molecule is a sequence that is substantially similar to the sequence of a native molecule. In the case of nucleotide sequences, variants include sequences that code for the same amino acid sequence as the native protein due to the degeneracy of the genetic code. Such natural allelic variants can be identified using molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those that code for native proteins and those that code for polypeptides with amino acid substitutions, created, for example, using site-directed mutagenesis. Generally, the nucleotide sequence variants of the present invention have at least 40%, 50%, 60% or 70%, such as 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%, typically at least 80%, such as 81%-84%, at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to the native (endogenous) nucleotide sequence. In certain embodiments, the variants are biologically functional (i.e., retain 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the activity or function of the wild-type).

[0253] A "conservative substitution" of a particular nucleic acid sequence refers to a nucleic acid sequence that codes for the same or essentially the same amino acid sequence. Because the genetic code is degenerate, any given polypeptide is encoded by a large number of functionally identical nucleic acids. For example, the codons CGT, CGC, CGA, CGG, AGA, and AGG all code for the amino acid arginine. Thus, wherever a codon specifies arginine, the codon can be changed to any of the corresponding codons described without changing the encoded protein. Such nucleic acid variations are "silent variations," which are a type of "conservatively modified variations." Any nucleic acid sequence described herein that codes for a polypeptide represents all possible silent variations, unless otherwise noted. Those skilled in the art will recognize that each codon in a nucleic acid (except ATG, which is usually the only methionine codon) can be modified by standard techniques to obtain a functionally identical molecule. Thus, each "silent variation" of a nucleic acid that codes for a polypeptide is implicit in each described sequence.

[0254] The term "substantial identity" of a polynucleotide sequence means that the polynucleotide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity when compared to a reference sequence by using one of the alignment programs described with standard parameters. It will be understood by those skilled in the art that these values ​​can be appropriately adapted to determine the corresponding identity of proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame position, etc. For these purposes, substantial amino acid sequence identity typically means at least 70%, at least 80%, 90% or even at least 95% sequence identity.

[0255] The term "substantial identity" with respect to a polypeptide indicates that the polypeptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98%, or 99% sequence identity to a reference sequence in a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunologically reactive with an antibody raised against the other polypeptide. Thus, a polypeptide is identical to another polypeptide, for example, when the only difference between the two polypeptides is a conservative substitution.

[0256] The terms "treat" and "treatment" refer to both therapeutic and prophylactic or preventative measures, the purpose of which is to prevent, inhibit, reduce, or diminish undesirable physiological changes or disorders, such as the onset, progression, or worsening of disorders. In the context of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization (i.e., no worsening or progression) of symptoms or adverse effects of disease, delay or slowing of disease progression, improvement or mitigation of disease state, and remission (whether partial or complete). "Treatment" can also mean a prolongation of survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder, as well as those with a predisposition (e.g., as determined by genetic assay).

[0257] The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0258] All methods and uses described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or illustrative phrases (such as "such as" or "for example") provided herein is intended only to better elucidate the invention and does not impose limitations on the scope of the invention unless otherwise stated. Nothing in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0259] All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced with an alternative feature that serves the same, equivalent or similar purpose. Thus, unless otherwise expressly stated, the disclosed features (e.g. modified nucleic acids, vectors, plasmids, recombinant vector sequences, vector genomes, or virus particles) are examples of the genus of equivalent or similar features.

[0260] As used herein, the forms "a," "and," and "the" include singular and plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to "a nucleic acid" includes a plurality of such nucleic acids, a reference to "a vector" includes a plurality of such vectors, and a reference to "a virus" or "AAV or rAAV particle" includes a plurality of such virions / AAV or rAAV particles.

[0261] As used herein, the term "about" refers to a value within 10% (plus or minus) of a reference value.

[0262] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within the range, unless otherwise stated herein, and each individual value is incorporated herein as if it were individually set forth herein.

[0263] Thus, all numerical values ​​or ranges include integers within such value ranges, and fractions of integers within those values ​​or ranges, unless the context clearly indicates otherwise. Thus, for example, 80% or greater identity includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., etc.

[0264] When referring to an integer with a more than or less than term, it includes all numbers greater than or less than that reference number. Thus, for example, less than 100 includes everything from 99, 98, 97, etc. through the number 1, and less than 10 includes everything from 9, 8, 7, etc. through the number 1.

[0265] As used herein, all numerical values ​​or ranges include fractions of those values ​​as well as integers within such ranges and fractions of integers within such ranges, unless the context clearly indicates otherwise. Thus, for example, a reference to a numerical range such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc. Thus, a reference to a range of 1-50 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc.

[0266] Reference to a series of ranges includes ranges combining the limits of the different ranges therein. Thus, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,500, 2,500-3,000, 3,000-3,500, 3,50 References to a series of ranges such as 0 to 4,000, 4,000 to 4,500, 4,500 to 5,000, 5,500 to 6,000, 6,000 to 7,000, 7,000 to 8,000, or 8,000 to 9,000 include ranges such as 10 to 20, 10 to 50, 30 to 50, 50 to 100, 100 to 300, 100 to 1,000, 1,000 to 3,000, 2,000 to 4,000, 4,000 to 6,000, etc.

[0267] VI. Kits The present invention provides a kit with packaging material and one or more components therein.The kit typically includes a label or package insert that includes a description of the components included therein or an instruction for the in vitro, in vivo, or ex vivo use of the components included therein.The kit can include a collection of such components, such as nucleic acids, recombinant vectors, viral particles, splicing modifier molecules, and optionally a second active agent, such as another compound, agent, drug, or composition.

[0268] A kit refers to a physical structure that contains one or more components of the kit. The packaging can maintain the components sterile and can be made of materials commonly used for such purposes (e.g., paper, cardboard, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0269] The label or package insert may include the identity of one or more components contained therein, dosage, clinical pharmacology of the active ingredient, such as mechanism of action, pharmacokinetics and pharmacodynamics. The label or package insert may include information identifying the manufacturer, lot number, place and date of manufacture, expiration date. The label or package insert may include information identifying the manufacturer information, lot number, place and date of manufacture. The label or package insert may include information regarding the disease for which the kit components may be used. The label or package insert may include instructions for the clinician or subject to use one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. The instructions may include dosage, frequency or duration of administration, and instructions for carrying out any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0270] The label or package insert may include information about benefits that the components may provide, such as preventive or therapeutic benefits. The label or package insert may include information about potentially harmful side effects, complications, or reactions, such as warnings to the subject or clinician about situations in which it may not be appropriate to use a particular composition. Since harmful side effects or complications may occur if the subject has previously taken, will take, or is currently taking one or more other medications that may be incompatible with the composition, or if the subject has previously taken, will take, or is currently taking another treatment protocol or therapeutic regimen that may be incompatible with the composition, the instructions may also include information about such incompatibilities.

[0271] A label or package insert includes "printed matter", such as paper or cardboard, either separate or attached to a component, kit or packaging material (e.g., a box), or attached to an ampoule, tube or vial that the kit component is in. The label or package insert can further include a computer readable medium, such as a printed label with a bar code, a disk, an optical disk, such as a CD- or DVD-ROM / RAM, a DVD, an MP3, or an electronic storage medium, such as RAM and ROM, or hybrids thereof, such as magnetic / optical storage media, flash memory, hybrids and memory type cards. EXAMPLES

[0272] VII. Working Examples Although several aspects of the present invention have been described, those skilled in the art may nonetheless make various modifications and changes to the present invention to adapt it to various usages and conditions without departing from the spirit and scope of the present invention. Thus, the following examples are intended to be illustrative and are not intended to limit the scope of the invention as claimed.

[0273] Example 1 Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by mutations in the SMN1 gene. The homologous SMN2 gene is unable to functionally compensate for SMN1 mutations because the majority of SMN2 transcripts skip exon 7, resulting in an unstable protein. 33 In the absence of SMN1, only 10% of the SMN2 protein is produced, which corresponds to the proportion of SMN2 transcripts that include exon 7. Modulation of the hypofunctional SMN2 "backup" gene by correcting exon 7 skipping of SMN2 is one of the most successful approaches to treat SMA.

[0274] Recently, Nusinersen (Ionis), an antisense oligonucleotide (ASO) that induces inclusion of SMN2 exon 7, became the first FDA-approved drug to treat SMA.34 LMI070 (SPINRAZA, Novartis 31 ) and RG7800 (Roche / PTC / SMAF 35 Small molecules that induce exon 7 inclusion, such as , are in clinical development.

[0275] LMI070 (Spinraza™, Novartis 31 ) has the following structure: TIFF2025060942000007.tif19128

[0276] RG7916 (Roche / PTC / SMAF 35 ) has the following structure: TIFF2025060942000008.tif30128

[0277] In one embodiment, drug-inducible SMN2 alternative splicing is used to control gene expression. In one aspect, the SMN2 minigene is fused to a cDNA encoding a mammalian transactivator, where the inclusion or exclusion of exon 7 determines the translation of the transactivator. The splicing modifier small molecule LMI070 can correct the skipping of exon 7 for transactivator expression, which then induces transcription from an optimized target gene expression cassette.

[0278] Example 2 We generated a chimeric SMN2 / transactivator minigene in which the production of the transactivator depends on the inclusion of SMN2 exon 7. The resulting transactivator binds to an optimized promoter and activates the expression of a downstream artificial miRNA (Figure 2). This system has 1) minimal miRNA expression in the off state, 2) significant induction of miRNA expression by the transactivator, 3) control of transactivator levels and subsequent miRNA expression levels, and 4) a size that allows packaging into a single recombinant adeno-associated virus (AAV). RNAi expression control will avoid sustained diversion of the RNAi pathway and minimize unintended chronic silencing of off-target genes.

[0279] Multiple DNA binding sites for a mammalian transactivator were cloned upstream of our optimized miRNA promoter. The transactivator is a fusion protein of a mammalian zinc finger protein engineered to bind to a specific DNA binding sequence and the Vp16 domain from herpes simplex virus. 36,37 Note that we chose the Vp16 domain instead of a strong activator domain (e.g., Vp64) to minimize possible gene activation as a result of off-target binding of the transactivator protein in the genome. Activation of the miRNA promoter was assessed by the luciferase ratio after triple transfection with the transactivator expression cassette, a miRNA promoter-driven firefly expression cassette, and a Renilla luciferase expression cassette (Figure 3a-b).

[0280] To regulate transactivator expression, the transactivator was cloned downstream of a self-cleaving 2A peptide and an SMN2 minigene containing exons 6-7 and the 5' end of exon 8, as well as the minimal intronic intervening sequence required to recapitulate SMN2 splicing. 38(Figure 4a). Note that 10% of the transcripts contain exon 7, similar to the native SMN2 gene state. This transcript produces a transactivator that partially activates the RNAi expression cassette. Therefore, the 3' and 5' exon 7 splice sites in the SMN2 / transactivator minigene were modified to either constitutively exclude exon 7 (CSI3, 3' modification) or to constitutively include exon 7 (CSI5, 5' modification). This minimizes background activation of the RNAi promoter. 39 (Figures 4b-c). Importantly, inclusion of exon 7 is LMI070 dose-responsive (Figures 5a-b). Also, the entire cassette fits within the rAAV.

[0281] We created non-allele-specific artificial miRNA sequences targeting either HTT exon 2 (mi2.4v1) or HTT exon 44 (miHDS1v6A). These miRNA sequences were engineered with siSPOTR to have limited off-target profiles. 40 In addition, we also designed specific seed-controlled miRNA sequences (mi2.4v1C and miHDS1v6a) to be used as controls. These miRNA controls do not silence HTT expression but contain the same miRNA seed (5' nucleotides 2-8) to correspond with the off-target profiles of mi2.4v1 and miHDS1v6a, respectively (Figure 6a-b).

[0282] Example 3 In vitro studies: Medium spiny neurons (MSNs) represent 95% of the neuronal population in the striatum, are the cell type most affected in HD, and are the main target cells in these studies. Therefore, MSNs were used in all in vitro studies. MSN cultures can be obtained by direct neurotransmission of human fibroblasts from healthy subjects or HD patients. 41,42 .

[0283] MSN cultures were transduced with rAAV2 / 1, an AAV serotype that efficiently transduces MSN neurons in vivo in mouse brain and in MSN cultures in vitro. 43 Previous reports have shown that reducing HTT expression by 50% in mouse brain is sufficient to ameliorate the disease phenotype. 8,11,12 Therefore, 50% silencing is the initial bar set for this regulated promoter system. MSN cultures are transduced with increasing doses of rAAV2 / 1 virus expressing mi2.4v1, and HTT mRNA levels are determined by Q-RTPCR after LMI070 treatment (1 μM). Mock-treated non-transduced and transduced MSN cultures are used as controls to define basal HTT expression levels, and to ensure that mi2.4v1 background levels do not appreciably silence HTT. Cells transduced with AAV2 / 1 expressing mi2.4v1 under the control of the U6 promoter are used as a positive silencing control. The aim is to define an AAV therapeutic window where HTT expression is reduced by more than 50% only in the presence of LMI070. Once an effective AAV dose has been established, the kinetics of the RNAi expression system in response to LMI070 will be analyzed by analyzing mi2.4v1 expression at different time intervals and at different LMI070 concentrations.

[0284] In the human brain, the top 20 endogenous miRNAs account for 75% of miRNA:mRNA binding sites. 44 By comparing the expression of mi2.4v1 with each of these top 20 miRNAs and their targeted mRNAs, endogenous pathway diversion is examined. Total RNA was extracted from LMI070- or mock-treated transduced MSN cultures, and mature miRNA levels were analyzed as previously performed. 45 , as determined by stem-loop Q-PCR. In the absence of LMI070, mi2.4v1 expression is not expected to interfere with endogenous RNAi regulation, as it is expressed in the presence of known endogenous target mRNAs. 44The mi2.4v1-associated off-target silencing is determined by RNA-seq using total RNA samples from transduced MSN cultures after LMI070 or mock treatment. In addition, transcriptome changes induced by LMI070 or the transactivator alone are examined. In these cases, transcriptome changes are determined using total RNA samples from MSN cultures treated with LMI070 and from transduced MSN cultures expressing only the transactivator protein. Given our initial data, LMI070-regulated miRNA expression is expected to confer at least 50% HTT silencing. Cytotoxicity - measured using nutrient withdrawal - is also expected to be minimal. 46 The lack of toxicity is also likely to correlate with minimal changes in cellular miRNA levels or target mRNA expression.

[0285] Example 4 In vivo regulation: rAAV2 / 1 virus expressing mi2.4v1 under the control of a regulated promoter system (rAAV.RPmi2.4v1) is injected into the striatum of N171-82Q transgenic mice, an established HD mouse model that expresses the first 171 amino acids of mutant Huntington protein in the mouse brain. 47First, to determine whether there is a dose at which the system is overloaded (too much background HTT silencing), rAAV.RPmi2.4v1 or rAAV.RPmi2.4v1C (seed-based control RNAi trigger) are given at 5E10, 2.5E10 and 5E9 vg / hemisphere (n = 10 male mice / group). As a control, mice are injected with rAAV viruses expressing mi2.4v1 or mi2.4v1C under the control of the U6 promoter, a strong Pol3 constitutive promoter. The aim is to determine the dose of AAV and LMI070 at which mi2.4v1 pulses induce mutant HTT suppression of 50% or more, and the time to reach peak suppression. Once the rAAV dose is determined, 3 weeks after delivery, 1–30 mg / kg LMI070 is administered by oral gavage. Note that 30 mg / kg is the maximum dose reported to increase SMN2 exon 7 inclusion in mouse brain, and 1 mg / kg is the minimum dose that has a therapeutic effect in the SMN mouse model. 31 Experimental controls are mice expressing mi2.4v1 or mi2.4v1C under regulated promoters, and mice expressing mi2.4v1 under the control of the mouse U6 promoter, both of which receive only the formulation buffer (mock treatment). Mice (n=8 male mice / group) are sacrificed 24, 48, 72, 96 and 120 hours after LMI070 (or mock) administration. Expression levels of mi2.4v1 and endogenous miRNAs as well as mutant HTT are assessed in brain lysates. This study provides relevant data on the efficacy of LMI070 to initiate an RNAi pulse and the expression kinetics of the system in vivo. The pharmacokinetics of LMI070 in mice orally administered with a dose of 3 mg / Kg had a Cmax (maximum concentration) of 86 nM and a Tmax (time to reach Cmax) of 4.3 hours in serum, with good distribution in the brain (brain:plasma ratio concentration of 1.4). Note that at this concentration (120 nM in brain), LMI070 induces inclusion of exon 7 with either the CSI or CSI3 minigene (Figure 5).

[0286] Data from previous studies will then be used to determine how often to pulse the HTT-targeting RNAi trigger for similar efficacy levels, as a guide for when re-administration may be necessary. Prior to injection, mice (n=15 male mice / group) were subjected to baseline rotarod testing to standardize groups, and then injected bilaterally with experimental or control AAV vectors ± LMI070. The accelerating rotarod provides a sensitive means to detect motor impairments in HD models. Following AAV injection, LMI070 is administered at a pre-established dose to reach ≥50% silencing over a 24-48 hour period, followed by re-administration once or twice weekly. At 10, 14, and 18 weeks, mice were subjected to additional rotarod testing as before. 11 The mice are then euthanized and the brains processed for RNA analysis, biochemical testing and histology.

[0287] The LMI070-induced RNAi pulse is expected to last for 24-48 hours and reduce mHTT levels by ≥50%. When LMI070 is removed, mi2.4v1 levels decline and mHTT levels are no longer suppressed (Figure 7). Previous studies using antisense oligonucleotide molecules (ASOs) have reported that reversal of disease phenotypes is longer-lasting than HTT silencing following transient ASO injections. 48Therefore, pulses of RNAi may need to be maintained for days or even weeks, similar to what was seen with ASOs targeting mHTT. Because exon 7 of SMN2 is included in 1% (CSI3) and 10% (CSI) of SMN2 / transactivator transcripts, the RNAi promoter may be partially activated even in the absence of LMI070. This may be problematic if the artificial miRNA levels are higher than the most abundant miRNA transcript levels. This problem may be solved by controlling transactivator expression using weaker promoters (e.g., pGK, mCMV). Previous studies have shown that LMI070 is not expected to be toxic to mice, but other small molecules that could be alternatives to LMI070 have been designed to induce inclusion of SMN2 exon 7 if observed. Because LMI070 can be administered orally and because it and some rAAV serotypes can cross the blood-brain barrier, this system also offers the potential to develop less invasive treatments for neurodegenerative brain diseases than current ASO therapies, which require multiple intrathecal injections.

[0288] Example 5 Previous studies using tissue samples from HD patients and mouse models have reported that transcriptome dysregulation is a key event occurring in the HD brain, occurring before cell loss is observed at early disease stages. 49~51 Recently, it has been reported that transcriptional changes in HD are not only restricted to the overall expression levels of specific genes but also to the transcript isoforms generated by alternative splicing. 32The goal is to identify alternative splicing changes that occur as a result of mutant HTT expression and are corrected in response to mutant HTT inhibition. This would provide an alternative autoregulatory alternative to the SMN2 exonic regulatory response elements, obviating the need for drug-related control. Alternative splicing events that change in response to mutant HTT expression hold promise as regulatory switches to control the production of transactivators in the context of HD. This work could provide the basis for using similar approaches to control expression in other neurodegenerative diseases.

[0289] Example 6 Alternative splicing events corrected by mutant HTT silencing: MSN cultures from HD human patients and controls are transduced with rAAV.U6mi2.4v1 or rAAV.U6miHDS1v6 to silence HTT expression, and rAAV.U6mi2.4v1C or rAAV.U6miHDS1v6aC as seed-matched controls (Figure 6). Note that by using two different miRNA sequences that effectively target HTT expression and their seed-matched miRNA controls, we will be able to distinguish between alternative splicing events due to mHTT silencing and events due to RNAi off-targeting.

[0290] One week after AAV transduction, when mHTT expression is reduced by 50%, cells are harvested and total RNA is extracted to determine alternative splicing changes. RNA-seq libraries are prepared using the TruSeq Stranded mRNA Sample prep kit (Illumina) and sent for sequencing using an Illumina HiSeq 4000. RNA-seq reads are mapped to the human genome (hg38) and transcriptome (Ensemble, release 89) using STAR software (2.5 or later), allowing up to three mismatches per read and up to 2 bp mismatches per 25 bp seed. Cuffdiff is used to calculate RNA-seq-based gene expression using the FPKM metric. 52,53 Using rMATS to identify differential alternative splicing events between sample groups corresponding to all five basic types of alternative splicing patterns 54 Significant splicing changes are considered using FDR<5% and ΔPSI≥5%. The top 10 alternative splicing events that show the greatest imbalance between transcript isoforms and are corrected upon silencing of mHTT protein are validated by PCR on independently obtained samples for biological confirmation.

[0291] Chimeric eGFP minigenes consisting of flanking alternatively spliced ​​exons and intervening minimal intron sequences are then generated and cloned upstream of eGFP cDNA in our AAV shuttle vector. These minigenes are designed to reduce eGFP expression after mHTT protein suppression to "normalize" the splicing profile to that of control MSN cultures. To test this system, MSN cultures are first transduced with rAAV2 / 1 expressing miRNAs targeting mutant HTT or seed-matched controls and rAAV expressing eGFP minigenes (or unmodified eGFP as a control). The kinetics of eGFP expression are determined by Western blot and quantitative fluorescence-based assays. Minigenes that respond accordingly to mHTT suppression are then used in place of the SMN2 minigene (Figure 2) and tested in Tg(N171-82Q) HD and zQ175 HD mouse models.

[0292] This disease-regulated expression system allows for the control of RNAi by exploiting alternative splicing changes that occur in response to mHTT expression. It is expected that a significant number of alternative splicing changes will be identified between HD and control MSN cultures. The alternative splicing changes are not expected to be limited to exon skipping or the other four types of alternative splicing patterns. It is expected that some events with a >2-fold imbalance between RNA transcript isoforms will be identified, and their ratios are expected to change upon silencing of mHTT protein. If no exon-skipping splicing events are found that fit the criteria of a >2-fold ratio, this can be resolved by introducing specific sequence modifications into the adjacent intron / exon constructs, similar to what was done with the SMN2 minigene, to improve the ratios close to but not exceeding those mentioned above. At the end of this study, we will have a set of disease-regulated minigenes that can be used to replace the SMN2 minigene.

[0293] Examples of differentially spliced ​​introns in HD include: TIFF2025060942000009.tif183150TIFF2025060942000010.tif231150TIFF2025060942000011.tif101150

[0294] Example 7 The following method was used to obtain the data illustrated in Figures 8-15. Briefly, Hek293 cells were treated with LMI070 at a concentration of 25 nM. RNA was extracted 12 hours after treatment using Trizol, followed by DNAseI treatment. Samples were assessed on an Agilent bioanalyzer for quality control, all samples had RIN values ​​>9.8, and were sent for Illumina RiboZero Gold total RNA library preparation and sequencing on an Illumina HiSeq4000. Illumina sequencing yielded 150 bp paired-end sequencing reads. Fastq files obtained from the Illumina platform were aligned to the human genome GRCh38 using the STAR aligner. The SAM format alignment file output from STAR was sorted, converted to BAM format, and indexed using Samtools. To visualize splicing in any region in the alignment, the BAM files were opened in IGV and visualized using the Sashimi plot function.

[0295] A custom R program was created to identify regions of interest that contained high levels of differential splicing. The input to this custom R program was the splice junction matrix output by STAR, ".SJ.out.tab". These files contained the raw counts obtained at each splice junction calculated during the alignment by STAR. This custom R program was created with the goal of identifying positions that are highly differentially spliced ​​between treated and control groups. The following steps were used to identify these regions: 1) Read the input file into R. 2) Create a unique position ID for each row (splice site) in the dataset. 3) Merge all samples into one data frame. 4) Calculate the mean count, sum of counts and standard deviation of counts for each splice site ID. 4) Replace NA (not available) values ​​with 0. 5) Extract all splice sites (rows) with sum=0 in the control. 6) Sort the extracted reads by mean count value from highest to lowest. This resulted in a list of splice sites with the highest levels of differential expression that had a sum of 0 counts in the controls.

[0296] The data in Figure 16 were obtained using the following method. The most relevant genes identified in the above analysis were validated by PCR. RNA was extracted from HEK293 cells treated with LMI070 (25 nM). PCR primers designed to bind upstream and downstream of the novel exons generated by LMI070 treatment were used. As observed by PCR, PCR products of increased size were detected in samples treated with LMI070 (Figure 16). These PCR products were sequenced using Sanger sequencing to confirm the inclusion of the identified novel exon sequences.

[0297] Example 8 The following method was used to obtain the data shown in Figure 19. HEK293 cells were transfected with a plasmid (0.3 μg) expressing the SMN2 minigene and treated 4 hours later with various doses of RG7800 (10 nM, 100 nM, 1 μM, and 10 μM). 24 hours after transfection, RNA was harvested, DNAseI treated, and 1 μg of RNA was reverse transcribed to assess SMN2 minigene splicing using PCR. PCR products were separated on a 3% agarose gel and exon 7 production was quantified using a ChemiDoc imaging system (Bio-Rad) and Imagine Lab analysis software.

[0298] The data shown in Figure 21 was obtained using the following method. HEK293 cells were transfected with a plasmid encoding the CRISPRi silencing system (dCas9 expression cassette, sgRNA expression cassette, and MCP-Krab expression cassette). Importantly, dCas9 was cloned under the SMN2 splice regulator cassette to control dCas9 protein expression with RG7800. 24 hours after transfection, cells were selected using puromycin (3 μM, 24 hours), passaged to a new plate, and treated with RG7800 (1 μM). 36 hours after RG7800 treatment, HEK293 cells were lysed using Passive lysis buffer (Promega, CA), and protein levels of Huntington (HTT), Cas9, and Beta cat were determined by Western blot. Beta catenin protein levels were determined as a loading control. HTT, Cas9 and Beta Cat protein levels were quantified using a ChemiDoc imaging system (Bio-Rad) and Imagine Lab analysis software.

[0299] All of the methods disclosed and claimed herein can be made and carried out without undue experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications may be made to the methods and the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutes and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined in the appended claims.

[0300] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2025060942000012.tif176151TIFF2025060942000013.tif216151TIFF2025060942000014.tif224150 TIFF2025060942000015.tif217151TIFF2025060942000016.tif224151TIFF2025060942000017.tif155151

Claims

1. 1. A medicament for treating a disease in a mammal comprising an expression cassette, the expression cassette being a first expression cassette comprising a chimeric gene operably linked to a first expression control element, the chimeric gene comprises a first portion comprising a minigene having an alternatively spliced ​​exon and a second portion encoding a first RNA encoding a protein, the first portion being 5' to the second portion, the second portion lacking an initiation codon or start codon, the alternatively spliced ​​exon comprising a translation initiation regulatory sequence, expression of the protein being controlled by inclusion of the alternatively spliced ​​exon, and the pharmaceutical agent being used in combination with a small molecule splicing modifier that modulates splicing of the alternatively spliced ​​exon; The small molecule splicing modifying agent is That is, The said medicine.

2. The pharmaceutical of claim 1, wherein the first RNA encoded by the second portion encodes a therapeutic protein.

3. The pharmaceutical of claim 2, wherein the therapeutic protein corrects a protein defect associated with a disease, thereby treating the disease.

4. The pharmaceutical of claim 1, wherein the protein is a transactivator protein.

5. The pharmaceutical of claim 4, further comprising a second expression cassette comprising a nucleic acid sequence encoding a second RNA, said nucleic acid sequence being operably linked to a second expression control element to which a transactivator protein binds, thereby increasing expression of said second RNA in a mammal and treating a disease.

6. The pharmaceutical of claim 4, which is used in combination with a second expression cassette comprising a nucleic acid sequence encoding a second RNA, the nucleic acid sequence being operably linked to a second expression control element to which a transactivator protein binds, thereby increasing expression of the second RNA in a mammal and treating a disease.

7. The pharmaceutical of claim 5 or 6, wherein the RNA encoded by the nucleic acid sequence of the second expression cassette is an inhibitory RNA.

8. The pharmaceutical according to claim 7, wherein the inhibitory RNA is an siRNA, an shRNA, or an miRNA.

9. The pharmaceutical of claim 7 or 8, wherein the inhibitory RNA inhibits or reduces expression of an abnormal protein or proteins associated with a disease, thereby treating the disease.

10. The pharmaceutical of any one of claims 1 to 9, wherein the first expression control element is a constitutive promoter, a cell type-specific promoter, or an inducible promoter.

11. The pharmaceutical of any one of claims 1 to 10, wherein the first part of the first expression cassette and the second part of the first expression cassette are separated by a cleavable peptide.

12. The pharmaceutical of claim 11, wherein the cleavable peptide is a self-cleaving peptide, a drug-sensitive protease, or a substrate for an endogenous endoprotease.

13. The pharmaceutical of any one of claims 1 to 12, wherein increased expression of the transactivator or protein encoded by the first RNA encoded by the second part of the first expression cassette is provided by inclusion of SMN2 exon 7.

14. The pharmaceutical of claim 13, wherein the inclusion of SMN2 exon 7 is triggered by the presence of a small molecule splicing modifier.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the first expression cassette or the second expression cassette is contained in a viral vector.

16. The pharmaceutical composition of claim 15, wherein the viral vector is selected from an adeno-associated viral (AAV) vector, a lentiviral vector, or a retroviral vector.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the disease is caused by a protein deficiency.

18. The method of any one of claims 1 to 16, wherein the disease is caused by a genetic defect.

19. The pharmaceutical composition according to any one of claims 1 to 16, wherein the disease is a neurodegenerative disease.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the mammal is a human.

21. A pharmaceutical composition according to any one of claims 1 to 20, for administration to the central nervous system or brain.

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