Compositions and Methods for Inducible Selective Splicing Regulation of Gene Expression

Abstract

A chimeric mini-gene is provided herein, and alternative splicing of the mini-gene determines whether the encoded gene is expressed. In particular, the mini-gene is alternatively spliced in response to a splicing modifying agent such that the encoded gene is expressed only in the presence of the splicing modifying agent. The encoded gene can encode an inhibitory RNA, a CRISPR-Cas9 protein, a transactivator, or a therapeutic protein.

Description

Technical Field

[0001] Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 343,381, filed May 18, 2022, the entire contents of which are incorporated herein by reference.

[0002] Reference to Sequence Listing This application contains a Sequence Listing XML, which is submitted electronically and is incorporated herein by reference in its entirety. The Sequence Listing XML created on May 11, 2023, is named CHOPP0056WO_ST26.xml and is 32,301 bytes in size.

[0003] 1. Field The present invention relates generally to the fields of molecular biology and medicine. More specifically, the present invention relates to compositions and methods for modulating the expression of therapeutic genes using alternative splicing regulation.

Background Art

[0004] 2. Description of Related Art Viral and non-viral approaches for gene therapy have advanced significantly over the past 20 years, but the main focus has been on cargo delivery systems; for example, the evolution and engineering of viral capsids for adeno-associated virus (AAV), the expansion of the outlook for cell-targeting envelopes for lentiviruses, and the improvement of lipid nanoparticles for improved uptake. However, the cargo itself, and more importantly the elements that control its expression, have been little touched except for using promoters or 3' regulatory elements engineered to restrict expression to certain cell types (Brown et al., 2006; Domenger & Grimm, 2019). Therefore, there is a need for compositions and methods for modulating the expression of therapeutic genes in cargo delivery systems.

Summary of the Invention

[0005] Summary Compositions and methods are provided herein for exquisitely controlling gene expression via a drug-inducible alternative splicing switch. Importantly, these compositions and methods do not require any bacterial or other external elements for regulation. These compositions and methods can be applied to any genetic element of interest in cells or animals, are orally bioavailable, and can utilize drugs used by humans.

[0006] In one aspect, a nucleic acid molecule comprising a first expression cassette comprising (a) a minigene having an exon that is alternatively spliced in the 5' to 3' direction and (b) a gene that is encoded, wherein exon 2 is the alternatively spliced exon, exon 2 comprises a translation initiation regulatory sequence, the minigene is derived from SF3B3, and the minigene comprises less than 700 base pairs, is provided herein. In some aspects, the translation initiation regulatory sequence in exon 2 comprises a start codon and a Kozak sequence. In some aspects, the nucleotide following the start codon in exon 2 is guanine.

[0007] In some aspects, intron 2 comprises the sequence of SEQ ID NO: 19, or a fragment or variant thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some aspects, exon 2 comprises the sequence of SEQ ID NO: 18, or a fragment or variant thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some aspects, intron 1 comprises the sequence of SEQ ID NO: 17, or a fragment or variant thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some aspects, the minigene comprises the sequence of SEQ ID NO: 1, or a fragment or variant thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0008] In some aspects, intron 2 comprises the sequence of SEQ ID NO: 25, or a fragment or variant thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some aspects, exon 2 comprises the sequence of SEQ ID NO: 24, or a fragment or variant thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some aspects, intron 1 comprises the sequence of SEQ ID NO: 23, or a fragment or variant thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some aspects, the minigene comprises the sequence of SEQ ID NO: 2, or a fragment or variant thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0009] In some aspects, the sequences of intron 1 and / or intron 2 do not contain any potential splice sites.

[0010] In some aspects, inclusion of exon 2 causes a frameshift. In some aspects, the number of nucleotides present in exon 2 is not divisible by 3. In some aspects, exon 3 contains a stop codon that is in-frame when exon 2 is skipped. In some aspects, the encoded gene is in-frame with the translation initiation regulatory sequence in exon 2.

[0011] In some instances, the encoded gene encodes a signal peptide such that the encoded protein enters the secretory pathway. In some instances, the amino acids encoded by exon 2 of the mini-gene correspond to the sequence of a predicted signal peptide. The sequence of the predicted signal peptide may correspond to the native signal peptide of the encoded gene or to a signal peptide that is heterologous to the encoded gene. In some instances, at least a portion of the native signal peptide of the encoded gene is deleted such that the resulting protein has a signal peptide that is partially encoded by exons 2 and 3 of the mini-gene and partially encoded by the encoded gene.

[0012] In some instances, the mini-gene comprises fewer than 600 or fewer than 500 nucleotides.

[0013] In some instances, the expression of the encoded gene does not require the co-expression of any exogenous regulatory protein. In some instances, the encoded gene encodes an inhibitory RNA, a therapeutic protein, a Cas9 protein, or a trans-activating factor protein. In some instances, the inhibitory RNA is an siRNA, shRNA, or miRNA. In some instances, the inhibitory RNA inhibits or reduces the expression of an abnormal or non-normal protein associated with a disease. In some instances, the therapeutic protein is a protein whose deficiency is associated with a disease. In some instances, what is encoded is not a reporter.

[0014] In some instances, the mini-gene and the encoded gene are separated by a cleavable peptide.

[0015] In some embodiments, the first expression cassette is operably linked to a first promoter. In some embodiments, the first promoter is a constitutive promoter. In some embodiments, the first promoter is the Rous sarcoma virus (RSV) promoter, the phosphoglycerate kinase (PGK) promoter, the JeT promoter, the CBA promoter, the synapsin promoter, or the minimal cytomegalovirus (mCMV) promoter.

[0016] In some embodiments, the nucleic acid molecule further comprises a second expression cassette. In some embodiments, the second expression cassette comprises a nucleic acid sequence encoding a guide RNA operably linked to a second promoter. In some embodiments, the second expression cassette comprises a nucleic acid sequence encoding a therapeutic protein, an inhibitory RNA, or a Cas9 protein, the nucleic acid sequence being operably linked to a second promoter, the second promoter being activated by a transactivator encoded by the first expression cassette.

[0017] In one aspect, cells comprising any one of the nucleic acid molecules of the embodiments of the present invention are provided herein.

[0018] In one aspect, a recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid protein and any one of the nucleic acid molecules of the embodiments of the present invention is provided herein.

[0019] In one aspect, a method for inducing the expression of a gene encoded in any one of the cells of the embodiments of the present invention, the method comprising contacting the cell with a splicing modifying agent, is provided herein. In some embodiments, in the presence of the splicing modifying agent, the second exon is included in the mRNA product of the nucleic acid, and in the absence of the splicing modifying agent, the exon is not included in the mRNA product of the nucleic acid. In some embodiments, the splicing modifying agent is LMI070 or RG7800 / RG7619.

[0020] In one aspect, provided herein is a method of administering a coded gene to a patient in need thereof, the method comprising administering to the patient any one of the nucleic acid molecules of the aspects of the present invention. In some aspects, the step of administering the coded gene comprises administering to the patient any one of the rAAVs of the aspects of the present invention.

[0021] In some aspects, the expression of the coded gene is regulated by cell type or tissue type. In some aspects, exon 2 is only included in the cell type or tissue type.

[0022] In some aspects, the method further comprises administering to the patient a splicing modifying agent to induce the expression of the coded gene. In some aspects, the splicing modifying agent is LMI070 or RG7800 / RG7619. In some aspects, the step of administering the splicing modifying agent is performed more than once. In some aspects, the step of administering the splicing modifying agent is performed at regular intervals. In some aspects, the step of administering the splicing modifying agent causes an increase in the expression of the coded gene, for example, by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, or 100-fold. In some aspects, the step of administering the splicing modifying agent causes an increase in the expression of the coded gene by at least 20-fold.

[0023] In some aspects, the rAAV vector comprises AAV particles containing the AAV capsid protein, and the first expression cassette and / or the second expression cassette is inserted between a pair of AAV inverted terminal repeats (ITRs). In some aspects, the rAAV is a self-complementary AAV (scAAV) vector. In some aspects, the rAAV is a single-stranded AAV (ssAAV). In some aspects, the AAV capsid protein is selected from the group consisting of, or derived from, 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, and AAV-2i8, or a capsid protein having at least 70% 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 some aspects, the pair of AAV ITRs is derived from, comprises, or consists of an ITR having at least 70% identity to the ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, or AAV-2i8, or the ITR sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-Rh10, or AAV-2i8.

[0024] In some aspects, multiple viral vectors are administered. In some aspects, the viral vector is administered at a dose of about 1×10 6 to about 1×10 18 vector genomes (vg / kg) per kilogram. In some aspects, the viral vector is about 1×10 per kilogram of patient7 ~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 about 1×10 13 ~1×10 14 are administered at a dosage of vg of about 1×10 6 ~1×10 16 particles vg / ml. In some scenarios, the viral vector is administered at a dosage of about 0.5 - 4 ml of 1×10

[0025] In some scenarios, the method further includes the step of administering a plurality of empty viral capsids. In some scenarios, the empty viral capsids are formulated with the viral particles to be administered to the patient. In some scenarios, the empty viral capsids are administered or formulated in a state where the viral vector particles or the empty viral capsids are in a 1.0 - 100-fold excess. In some scenarios, the empty viral capsids are administered or formulated in a state where the viral vector particles are in a 1.0 - 100-fold excess relative to the empty viral capsids. In some scenarios, the empty viral capsids are administered or formulated in a state where the empty viral capsids are about 1.0 - 100-fold in excess relative to the viral vector particles.

[0026] In some aspects, administration is to the central nervous system. In some aspects, administration is to the brain. In some aspects, administration is to the cisterna magna, the intracerebroventricular space, the ependyma, the ventricle, the subarachnoid space, and / or the intramedullary cavity. In some aspects, the ventricle is the rostral lateral ventricle, and / or the caudal lateral ventricle, and / or the right ventricle, and / or the left 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. In some aspects, the step of administering comprises intracerebroventricular injection and / or parenchymal injection. In some aspects, administration is at a single location within the brain. In some aspects, administration is at 1 to 5 locations within the brain.

[0027] In some aspects, the patient is human.

[0028] In some aspects, the method further comprises the step of administering one or more immunosuppressive agents. In some aspects, the immunosuppressive agent is administered before or simultaneously with the administration of the expression cassette. In some aspects, the immunosuppressive agent is an anti-inflammatory agent.

[0029] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, various modifications and variations within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description, and thus, the detailed description and specific examples, while indicating preferred embodiments of the present invention, are to be construed as merely illustrative.

Brief Description of the Drawings

[0030] The accompanying drawings, which form a part of this specification, are included to further illustrate certain aspects of the present invention. The present invention can be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

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Mode for Carrying Out the Invention

[0031] Detailed Description To date, gene therapy for human application has relied on engineered promoters that cannot be precisely controlled. Optimized switch elements are provided herein that allow for precise control of gene silencing or gene replacement after exposure to small molecules. Importantly, these small molecule inducers are used in humans, are orally bioavailable when administered to animals or humans, and can reach both peripheral tissues and the brain. Furthermore, the optimized switch system (miniX on ) does not require the co-expression of any regulatory proteins. Using miniX on , translation of the desired element for gene knockdown or gene replacement occurs after a single oral administration, and the expression level can be controlled by drug dose or by waves of repeated drug intake. This optimized switch can provide transient control of gene editing machinery and gene addition cassettes that can be adapted for cell biology applications and animal studies. Furthermore, due to the oral bioavailability and safety of the drugs used, the miniX on switch provides an unprecedented opportunity to improve gene therapy for more appropriate human applications.

[0032] I. Transgene Expression Regulated by Alternative Splicing Chimeric mini genes are disclosed herein, and alternative splicing of the mini genes determines whether the downstream-encoded gene is expressed. The encoded gene can be an inhibitory RNA, a CRISPR-Cas9 protein, a therapeutic protein, or a transactivator.

[0033] In one example, the minigene includes three exons, exons 1-3, and exon 2 is skipped in the basal state. Since the translation initiation regulatory sequence is located in exon 2, when exon 2 is skipped, the downstream encoded gene is not produced. Accordingly, translation of the encoded protein is not initiated. To turn on the expression of the encoded gene, 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, the minigene may include upstream and downstream exons from SF3B3, in addition to an intervening pseudoexon, where the pseudoexon is skipped in the basal state. However, in the presence of certain small molecule splicing modifiers (e.g., LMI070 or RG7800 / RG7619), the pseudoexon is included. Accordingly, the downstream encoded gene will be expressed in the presence of LMI070 or RG7800 / RG7619, but not in their absence.

[0034] Expression of the chimeric minigene can be regulated by various types of promoters, depending on the desired expression pattern. For example, the promoter can be a ubiquitously 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 for neuronal expression. As yet another example, the promoter can be an inducible promoter.

[0035] The chimeric mini-gene may have a cleavable peptide located between the mini-gene and the encoded gene. 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. When this peptide is present, the peptide encoded by the mini-gene will be separated from the encoded protein after translation. Optionally, the cleavable peptide may be a cleavage site of a widely expressed endogenous endoprotease, such as, for example, furin, prohormone convertase 7 (PC7), paired basic amino-acid cleaving enzyme 4 (PACE4), or subtilisin kexin isozyme 2 (SKI-1). Optionally, the cleavable peptide may be a cleavage site of a tissue-specific endoprotease or a cell-specific endoprotease (e.g., prohormone convertase 2 (PC2; mainly expressed in endocrine tissues and the brain), prohormone convertase 1 / 3 (PC1 / 3; mainly expressed in endocrine tissues and the brain), prohormone convertase 4 (PC4; mainly expressed in the testis and ovary), and proprotein convertase subtilisin kexin 9 (PSCK9; mainly expressed in the lung and liver), etc.).

[0036] II. Target Genes for Alternative Splicing Regulation A. Inhibitory RNA "RNA interference (RNAi)" is a 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 (in the case of Huntington's disease), SCA (in the case of spinocerebellar ataxia (types 1, 2, 3, 6, 7)), FXTAS (in the case of fragile X tremor / ataxia syndrome), and FMRP (in the case of fragile X).

[0037] "Interfering RNA", "RNAi", "small interfering RNA", "short interfering RNA" or "siRNA" molecules, "short hairpin RNA" or "shRNA" molecules, or "miRNA" are RNA duplexes of nucleotides that target nucleic acid sequences of interest. As used herein, the term "siRNA" is a generic name that encompasses subsets of shRNA and miRNA. "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule. Since the nucleotide sequence of the duplex portion of siRNA is complementary to the nucleotide sequence of the target gene, siRNA is "targeted" to that gene. In certain embodiments, siRNA is targeted to the sequence encoding Huntington. In some embodiments, the length of the siRNA duplex 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 in length. In certain embodiments, the length of the duplex is 19 base pairs or 21 base pairs in length. The RNA duplex portion of 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 that form 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 in length. In certain embodiments, the loop is 18 nucleotides in length. 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 in length.

[0038] shRNA is composed of a stem-loop structure designed to contain a 5' adjacent region, an siRNA region segment, a loop region, a 3' siRNA region, and a 3' adjacent region. In most RNAi expression strategies, short hairpin RNAs (shRNAs) driven by strong polIII promoters have been utilized. Although many shRNAs show effective knockdown of target sequences both in vitro and in vivo, some shRNAs that show effective knockdown of target genes have also been found to be toxic in vivo.

[0039] miRNAs are small cellular RNAs (about 22 nt) processed from precursor stem-loop transcripts. Known miRNA stem-loops can be modified to contain RNAi sequences specific to the gene of interest. miRNA molecules may be preferred over shRNA molecules because miRNAs are endogenously expressed. Therefore, miRNA molecules are less likely to induce the dsRNA-responsive interferon pathway, are processed more efficiently than shRNAs, and have been shown to silence 80% more effectively than shRNAs.

[0040] A recently discovered alternative approach is the use of artificial miRNAs (pri-miRNA scaffolds that shuttle siRNA sequences) as RNAi vectors. Artificial miRNAs are more similar to endogenous RNAi substrates and are also more suitable for Pol-II transcription (e.g., enabling tissue-specific expression of RNAi) and polycistronic strategies (e.g., enabling delivery of multiple siRNA sequences). See U.S. Patent No. 10,093,927, which is incorporated herein by reference.

[0041] The transcription unit of 「shRNA」 is composed of a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides. shRNA is exported from the nucleus by exportin-5 and, upon entering the cytoplasm, undergoes processing by Dicer to generate functional siRNA. The stem-loop of 「miRNA」 is composed of a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides, which is typically expressed as part of a larger primary transcript (pri-miRNA), and it is excised by the Drosha-DGCR8 complex to generate an intermediate known as pre-miRNA, which is then exported from the nucleus by exportin-5 and, upon entering the cytoplasm, undergoes processing by Dicer to generate functional siRNA. 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 to 20 nucleotides) excised by Drosha and Dicer processing has been replaced with an siRNA sequence for the target gene while maintaining the structural elements within the stem loop required for efficient Drosha processing. The term 「artificial」 is derived 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 natural miRNA sequences and artificially created miRNA shuttle vectors.

[0042] siRNA can be encoded by a nucleic acid sequence, which 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 Ts.

[0043] In the design of RNAi, there are several factors that need to be considered, such as the properties of siRNA, the persistence of the silencing effect, and the selection of the delivery system. To produce the RNAi effect, the siRNA introduced into an organism will typically contain an exonic sequence. Furthermore, since the RNAi process is homology-dependent, the sequence must be carefully selected to maximize gene specificity while minimizing the potential for cross-interference between sequences that are homologous but not gene-specific. Preferably, the siRNA exhibits an identity greater than 80%, 85%, 90%, 95% or 98%, and even 100% identity between the sequence of the siRNA and the gene to be inhibited. Sequences with an identity of less than about 80% to the target gene are significantly less effective. Thus, the higher the homology between the siRNA and the gene to be inhibited, the lower the likelihood that the expression of unrelated genes will be affected.

[0044] In addition, the size of the siRNA is also an important consideration. In some embodiments, the present invention relates to siRNA molecules that comprise at least about 19 to 25 nucleotides and are capable of modulating gene expression. For the purposes of the present invention, the siRNA is preferably less than 500, 200, 100, 50 or 25 nucleotides in length. More preferably the siRNA is from about 19 nucleotides to about 25 nucleotides in length.

[0045] An siRNA target generally means a polynucleotide comprising a region encoding a polypeptide, or a polynucleotide comprising a polynucleotide region that regulates other processes important for replication, transcription or translation or the expression of a polypeptide, or a polynucleotide comprising both a region encoding a polypeptide and a region functionally linked thereto that regulates expression. Any gene expressed in a cell can be targeted. Preferably, the target gene is involved or associated with the progression of a cellular activity important for a disease or a cellular activity of particular interest as a research subject.

[0046] B. CRISPR System Gene editing is a technology that enables the modification of target genes in living cells. In recent years, the implementation of on-demand gene editing using the bacterial CRISPR immune system has brought about a major transformation in the way scientists approach genome editing. The Cas9 protein of the CRISPR system, an RNA-guided DNA endonuclease, can be engineered to target new sites relatively easily by changing its guide RNA sequence. This discovery has made sequence-specific gene editing functionally effective.

[0047] Generally, the term "CRISPR system" collectively refers to transcripts and other elements involved in the expression or direction of activity of CRISPR-associated ("Cas") genes, such as sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or the active partial tracrRNA), tracr-mate sequences (including "direct repeats" and, in the case of endogenous CRISPR systems, the partial direct repeats that have undergone tracrRNA processing), guide sequences (also called "spacers" in the case of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus. Examples of genes whose expression can be inhibited or whose sequences can be edited using the CRISPR expression system of the present disclosure include, but are not limited to, HTT (in the case of Huntington's disease), SCA (in the case of spinocerebellar ataxia (types 1, 2, 3, 6, 7)), FXTAS (in the case of fragile X-associated tremor / ataxia syndrome), and FMRP (in the case of fragile X).

[0048] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that specifically binds to DNA and a Cas protein (such as Cas9) having nuclease functionality (e.g., two nuclease domains). The CRISPR / Cas system is classified into two classes, including six types and numerous subtypes. The classification is ultimately based on identifying all cas genes at the CRISPR / Cas locus and determining the signature genes at each CRISPR / Cas locus, and can place the CRISPR / Cas system into either class 1 or class 2 based on the effector module, i.e., the genes encoding the proteins involved in the interference stage. Class 1 systems have a multi-subunit crRNA-effector complex, whereas class 2 systems have a single protein such as Cas9, Cpf1, C2c1, C2c2, C2c3, or a crRNA-effector complex. Class 1 systems include type I, type III, and type IV systems. Class 2 systems include type II, type V, and type VI systems. Thus, one or more elements of the CRISPR system can be derived from any class or type of the CRISPR system, and can be derived from a particular organism, including an endogenous CRISPR system such as Streptococcus pyogenes.

[0049] As discussed herein, the CRISPR system can induce double stranded breaks (DSBs) at target sites, followed by fragmentation. In another aspect, a Cas9 variant regarded as a "nickase" is used to nick a single strand at the target site. For example, for purposes such as improving specificity, a pair of nickases each directed by a different gRNA that targets a sequence such that a 5' overhang is introduced when the nicks are introduced simultaneously can be used. In another aspect, 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 includes a transcriptional repressor or transcriptional activator fused to a ribosome-binding protein.

[0050] In some scenarios, Cas nuclease and gRNA (including a fusion of a crRNA specific to the target sequence and an invariant tracrRNA) are introduced into cells. Generally, the target site at the 5' end of the gRNA targets the Cas nuclease to the target site, such as a gene, using complementary base pairing. The target site can be selected based on its position immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG or NAG. In this regard, the 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. Generally, the CRISPR system is characterized by elements that facilitate the formation of the CRISPR complex at the site of the target sequence. Typically, a "target sequence" generally refers to a sequence designed such that the guide sequence has complementarity thereto, and hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. Full complementarity is not necessarily required if there is sufficient complementarity to cause hybridization and facilitate the formation of the CRISPR complex.

[0051] The target sequence can include any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. The target sequence can be located within the nucleus or cytoplasm of a cell, such as within an organelle of the cell. Generally, a sequence or template that can be used for recombination into a target locus containing the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some aspects, an exogenous template polynucleotide can be referred to as an editing template. In some aspects, the recombination is homologous recombination.

[0052] Typically, in the case of an endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence that hybridizes to a target sequence and forms a complex 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). A tracr sequence that can include or consist of all or part of a 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) can also form part of a CRISPR complex, such as by hybridization to all or part of a 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 the CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity over the full length of the tracr mate sequence when optimally aligned.

[0053] One or more vectors that drive the expression of one or more elements of the CRISPR system can be introduced into a cell such that the expression of those elements of the CRISPR system directs the formation of CRISPR complexes at one or more target sites. The components can also be delivered to the cell as proteins and / or RNAs. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. The Cas enzyme can be a target gene that is subject to the control of the regulatory alternative splicing events 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.

[0054] Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in one vector, and one or more additional vectors can provide any components of the CRISPR system not included in the first vector. The vector can 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, CRISPR activity can be targeted to multiple corresponding target sequences within a cell using a single expression construct.

[0055] The vector can include a regulatory element operably linked to an enzyme coding sequence encoding 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, their homologs, or modified forms thereof. These enzymes are known. For example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0056] The CRISPR enzyme can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae). The CRISPR enzyme can direct cleavage of one or both strands at the location of a 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 has been mutated such that, compared to the corresponding wild-type enzyme, it lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, a substitution of aspartic acid to alanine (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 that cleaves one strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence, e.g., in combination with two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination allows for nicking both strands and using them for the induction of NHEJ or HDR.

[0057] 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 can be of or derived from a particular organism, such as, but not limited to, a mammal including a human, mouse, rat, rabbit, dog, or non-human primate. Generally, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon of a native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence, in order to enhance expression in the host cell of interest. Different species exhibit a particular bias for a particular codon of a particular amino acid. Codon bias (the difference in codon usage frequency between organisms) often correlates with the translation efficiency of messenger RNA (mRNA), and it is thought to depend, inter alia, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of a selected tRNA in a cell is generally a reflection of the codons that are most frequently used in peptide synthesis. Thus, genes can be adapted for optimal gene expression in a given organism based on codon optimization.

[0058] Generally, a guide sequence is any polynucleotide sequence having complementarity with a target polynucleotide sequence that is sufficient to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the 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 an appropriate alignment algorithm.

[0059] Optimal alignment can be determined using any suitable algorithm for aligning sequences, and non-limiting examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, California), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0060] The CRISPR enzyme may be part of a fusion protein that includes one or more heterologous protein domains. The CRISPR enzyme fusion protein can include any additional protein sequence and, optionally, a linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional 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) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. 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 blue fluorescent protein (BFP), including autofluorescent proteins. The CRISPR enzyme can be fused to a gene sequence that encodes a protein or a fragment of such a protein that binds to a DNA molecule or other cellular molecule, 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 a fusion protein containing the CRISPR enzyme are described in US 20110059502, which is incorporated herein by reference.

[0061] C. Therapeutic Proteins Some aspects 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; in the case of STXBP1 deficiency, a form of neonatal epilepsy, a form of developmental delay), SCN1a (also known as hereditary epileptic encephalopathy, in the case of Dravet syndrome, also known as severe myoclonic epilepsy in infancy (SMEI); mutations in Nav1.1); SCN1b (mutations in the Nav1.1β subunit); SCN2b (in the case of familial atrial fibrillation; the β2 subunit of the type II voltage-gated sodium channel); KCNA1 (in the case of autosomal dominant episodic ataxia; muscle spasms with or without ataxia); KCNQ2 (KCNQ2-related epilepsy); GABRB3 (early-onset epilepsy; the β3 subunit of the GABAA receptor); CACNA1A (in the case of familial ataxia and hemiplegic migraine; the transmembrane pore-forming subunit of the P / Q-type voltage-gated calcium channel); CHRNA2 (in the case of autosomal dominant nocturnal frontal lobe epilepsy; the α subunit of the neuronal nicotinic acetylcholine receptor (nAChR)); KCNT1 (in the case of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and malignant migrating partial seizures in infancy (MMPSI); sodium-activated potassium channel); SCN8A (in the case of epilepsy and neurodevelopmental disorders; Nav1.6 deficiency, voltage-dependent sodium channel); CHRNA4 - α subunit (in the case of autosomal dominant nocturnal frontal lobe epilepsy; mutations in the α subunit of the nicotinic acetylcholine receptor); CHRNB2 - b2 subunit (in the case of autosomal dominant nocturnal frontal lobe epilepsy; mutations in the α subunit of the nicotinic acetylcholine receptor); ARX (in the case of Otohara syndrome, polyAla expansion in the ARX gene); MECP2 (in the case of Rett syndrome); FMRP (in the case of fragile X); and CLN3 (in the case of CLN disease, also known as juvenile Batten disease and also known as JNCL), but are not limited thereto.Other examples of therapeutic proteins that can be expressed using the expression system of the present disclosure include erythropoietin (EPO, in the case of anemia), progranulin (GRN, in the case of neurodegenerative diseases), tripeptidyl peptidase 1 (TPP1, in the case of lysosomal storage diseases), factor IX (F9, in the case of hemophilia), human α-galactosidase (GLA, in the case of Fabry disease), α-1-antitrypsin (A1AT, in the case of α-1-antitrypsin deficiency), human growth hormone (HGH, in the case of growth hormone deficiency), ion channels, components of the complement pathway, cytokines, chemokines, chemoattractants, protein hormones (e.g., EGF, PDF), protein components of serum, antibodies, secreted toll-like receptors, coagulation factors, kinase growth factors, and other signaling molecules. Other examples of proteins that can be expressed using the expression system of the present disclosure can be found in Lindy et al. (2018) and Heyne et al. (2018), as well as U.S. Patent Application Publication No. 2018 / 0353616, each of which is incorporated herein by reference in its entirety.

[0062] Disorders for which the present invention is useful include Pompe disease, Gaucher disease, beta-thalassemia, Huntington's disease; Parkinson's disease; muscular dystrophy (e.g., Duchenne and Becker types, etc.); hemophilia disorders (e.g., hemophilia B (FIX), hemophilia A (FVIII), etc.); SMN1-related spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-related galactosemia; cystic fibrosis (CF); SLC3A1-related disorders including cystinuria; COL4A5-related disorders including Alport syndrome; galactocerebrosidase deficiency; X-linked adrenoleukodystrophy and adrenomyeloneuropathy; Friedreich's ataxia; Pelizaeus-Merzbacher disease; TSC1 and TSC2-related tuberous sclerosis; Sanfilippo B syndrome (MPS IIIB); CTNS-related cystinosis; FMR1-related disorders including fragile X syndrome, fragile X-associated tremor / ataxia syndrome, and fragile X premature ovarian insufficiency syndrome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia (AT); Niemann-Pick disease type C1; juvenile neuronal ceroid lipofuscinosis (JNCL), juvenile Batten disease, Santavuori-Haltia disease, Jansky-Bielschowsky disease, and neuronal ceroid lipofuscinosis-related diseases including PPT-1 and TPP1 deficiencies; EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5-related childhood ataxia with central nervous system hypomyelination / leukodystrophy; CACNA1A and CACNB4-related episodic ataxia type 2; MECP2-related disorders including classical Rett syndrome, MECP2-related severe neonatal encephalopathy, and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy disease (SBMA); autosomal dominant cerebral arteriopathy with subcortical infarcts and leukoencephalopathy associated with Notch-3 (CADASIL); SCN1A and SCN1B-related seizure disorders; Alpers-Huttenlocher syndrome, POLG-related sensory ataxic neuropathy, dysarthria, and ophthalmoparesis, and polymerase G-related disorders including autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; Wilson's disease;Disorders such as Fabry disease are included, but are not limited thereto.;

[0063] In some aspects, the protein or polypeptide can be modified to increase stability in serum. Thus, when the present application refers to the function or activity of a "modified protein" or "modified polypeptide", it will be understood by those skilled in the art that it includes a protein or polypeptide that has additional advantages, for example, compared to an unmodified protein or polypeptide. It is particularly contemplated that aspects regarding "modified proteins" can also be practiced with respect to "modified polypeptides", and vice versa.

[0064] Recombinant proteins can have amino acid deletions and / or substitutions. Thus, proteins with deletions, proteins with substitutions, and proteins with both deletions and substitutions are modified proteins. In some embodiments, these proteins can further contain inserted or added amino acids, such as, for example, fusion proteins or proteins with linkers. A "modified deletion protein" lacks one or more residues of the native protein, but can have the specificity and / or activity of the native protein. A "modified deletion protein" can also have reduced immunogenicity or antigenicity. Examples of modified deletion proteins are those in which amino acid residues are deleted from at least one antigenic region, i.e., a region of the protein determined to be antigenic in a particular organism, for example, the type of organism in which the modified protein can be administered.

[0065] A substitution variant or replacement variant typically contains an exchange of one or more amino acids for another at one or more sites within a protein and can be designed to modulate one or more properties of the polypeptide, particularly its effector function and / or bioavailability. The substitution may be a conservative substitution, i.e., one in which an amino acid is replaced with one having a similar shape and charge, or it may not be. Conservative substitutions are well known in the art and include, for example, the change from alanine to serine, from arginine to lysine, from asparagine to glutamine or histidine, from aspartic acid to glutamic acid, from cysteine to serine, from glutamine to asparagine, from glutamic acid to aspartic acid, from glycine to proline, from histidine to asparagine or glutamine, from isoleucine to leucine or valine, from leucine to valine or isoleucine, from lysine to arginine, from methionine to leucine or isoleucine, from phenylalanine to tyrosine, leucine or methionine, from serine to threonine, from threonine to serine, from tryptophan to tyrosine, from tyrosine to tryptophan or phenylalanine, and from valine to isoleucine or leucine.

[0066] In addition to deletions or substitutions, the modified protein may have an insertion of a residue. This typically involves the addition of at least one residue in the polypeptide. This can include the insertion of a targeting peptide or targeting polypeptide or just a single residue insertion. Terminal additions, called fusion proteins, will be described later.

[0067] The term "biologically functional equivalent" is well understood in the art and is further detailed 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 the amino acids of a control polypeptide are included as long as the biological activity of the protein is maintained. A recombinant protein can be a biologically functional equivalent to the corresponding native protein in certain aspects.

[0068] It will also be understood that amino acid and nucleic acid sequences can include additional residues, such as additional N-terminal or C-terminal amino acids, or 5' or 3' sequences, and still be essentially as described in one of the sequences disclosed herein as long as the sequence meets the above criteria, including maintenance of biological protein activity where protein expression is involved. The addition of terminal sequences is particularly applicable to nucleic acid sequences that can include various non-coding sequences adjacent to either the 5' or 3' portion of the coding region, or various internal sequences known to be present within a gene, i.e., introns.

[0069] As used herein, a protein or peptide generally refers to a protein translated from a gene that is more than about 200 amino acids up to the full-length sequence at most; a polypeptide that is more than about 100 amino acids; and / or a peptide that is about 3 to about 100 amino acids, but is not limited thereto. For convenience, the terms "protein", "polypeptide" and "peptide" are used interchangeably herein.

[0070] As used herein, "amino acid residue" refers to any natural 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 continuous and have no non-amino acid sequences interrupting the sequence of amino acid residues. In another embodiment, the sequence may contain 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.

[0071] Accordingly, the term "protein or peptide" encompasses an amino acid sequence that includes at least one of the 20 common amino acids found in natural proteins, or at least one modified or unnatural amino acid.

[0072] Certain embodiments of the invention relate to fusion proteins. These molecules can have a therapeutic protein linked to a heterologous domain at the N-terminus or C-terminus. For example, a leader sequence from another species can also be used in the fusion to enable recombinant expression of the protein in a heterologous host. Other useful fusions include the addition of a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, preferably cleavable, 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).

[0073] Methods for making fusion proteins are well known to those of skill in the art. Such proteins can be produced, for example, by de novo synthesis of the complete fusion protein or by attachment of a DNA sequence encoding the heterologous domain followed by expression of the intact fusion protein.

[0074] The production of a fusion protein that restores the functional activity of a parent protein can be facilitated by linking genes with a bridging DNA segment encoding a peptide linker that is joined between tandemly linked polypeptides. The linker will be of sufficient length to allow proper folding of the resulting fusion protein.

[0075] III. Splicing modifiers A representative splice modifier is LMI070 (5-(1H-pyrazol-4-yl)-2-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)phenol; Spinraza™; Novartis 31 ) which can penetrate the blood-brain barrier and has the following structure: TIFF2025521120000001.tif18128.

[0076] Examples of alternative splicing events that are only included in the presence of LMI070 and can be used for the control of gene expression in the systems of the present disclosure include, but are not limited to, 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 (chr12:4,536,017~4,538,508), SSBP1 (chr7:141,739,167~141,742,229), RARS (chr5:168,517,815~168,519,190), PDXDC2P (chr16:70,030,988~70,031,968), STRADB (chr2:201,469,953~201,473,076), WNK1 (chr12:894,562~896,732), WDR27 (chr6:169,660,663~169,662,424), CIP2A (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 (chr21:37,420,144~37,473,056), 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).In the presence of LMI070, the inclusion of novel exons is enhanced, and examples of alternative splicing events that can be used for the control of gene expression in the systems of the present disclosure 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 - 130,167,688), 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 (chr13:72,718,939 - 72,727,611), LINC01296 (chr14:19,092,877 - 19,096,652), SF3B3 (chr16:70,541,627 - 70,544,553), SAFB (chr19:5,654,060 - 5,654,457), GCFC2 (chr2:75,702,163 - 75,706,652), MRPL45 (chr17:38,306,450 - 38,319,088), SPIDR (chr8:47,260,788 - 47,280,196), 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), but are not limited thereto. For the above list, each genomic position includes the upstream and downstream exons and intervening intron sequences targeted by LMI070.

[0077] For example, the following are included, which are analogs of splice modifiers such as LMI070 and can be used similarly: 6-(naphthalen-2-yl)-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine, 6-(benzo[b]thiophen-2-yl)-N-methyl-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine, 2-(6-(2,2,6,6-tetramethylpiperidin-4-ylamino)pyridazin-3-yl)phenol, 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-tetramethylpiperidin-4-yl)pyridazin-3-amine, 3-(benzo[b]thiophen-2-yl)-6-(2,2,6,6-tetramethylpiperidin-4-yloxy)pyridazin, 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-(quinolin-7-yl)-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine, 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 - pyrazol - 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]oxadiazol - 5 - yl) - N - (2,2,6,6 - tetramethyl - piperidin - 4 - yl)pyridazin - 3 - amine, 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, 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]thiophen-2-yl)-N-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyridazin-3-amine, 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, 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-(pyrrolidin-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]nona-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]-pyridazin-3-yl}-5-pyrazol-1-yl-phenol, 2-[6-(3,6-diaza-bicyclo[3.2.1]octa-3-yl)-pyridazin-3-yl]-5-pyrazol-1-yl-phenol, 2-[6-(2,7-diaza-spiro[3.5]nona-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]nona-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-tetra-hydro-pyridin-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-tetramethyl-1,2,3,6-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)amino)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-yl-oxy)-propyl]-carbamate, tert-Butyl minate, 7-(3-amino-propoxy)-3-{6-[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]-pyridazin-3-yl}-naphthalene-2-ol, N-[3-(7-hydroxy-6-{6[methyl-(2,2,6,6-tetramethyl-piperidin-4-yl)-amino]-pyridazin-3-yl}-naphthalene-2-yloxy)-propyl]-acetamide, 7-(3-hydroxypropoxy)-3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalene-2-ol, 7-(3-methoxypropoxy)-3-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)naphthalene-2-ol, 7-(2-morpholinoethoxy)-3-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)pyridazin-3-yl)naphthalene-2-ol, 3-(6-(piperidin-4-ylmethyl)pyridazin-3-yl)naphthalene-2-ol, 5-(1H-pyrazol-1-yl)-2-(6-((2,2,6,6-tetramethylpiperidin-4-yl)methyl)pyridazin-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)naphthalene - 2 - ol, 3 - (6 - (methyl(2,2,6,6 - tetramethylpiperidin - 4 - yl)amino)pyridazin - 3 - yl)-7 - (tetrahydro - 2H - pyran - 4 - yl)naphthalene - 2 - ol, 7 - (difluoromethyl)-3 - (6 - (methyl(2,2,6,6 - tetramethylpiperidin - 4 - yl)amino)pyridazin - 3 - yl)naphthalene - 2 - ol, 7 - ((4 - hydroxy - 2 - methylbutan - 2 - yl)oxy)-3 - (6 - (methyl(2,2,6,6 - tetramethylpiperidin - 4 - yl)amino)pyridazin - 3 - yl)naphthalene - 2 - ol, 7 - (3 - hydroxy - 3 - methylbutoxy)-3 - (6 - (methyl(2,2,6,6 - tetramethylpiperidin - 4 - yl)amino)pyridazin - 3 - yl)naphthalene - 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)-5 - (1H - pyrazol - 4 - yl)phenol, 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)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)-5-(5-methyloxazol-2-yl)phenol, 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)phenol, 4-(1H-indol-2-yl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol, 4-(cyclopenta-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(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridin-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-tetramethylpiperidin-4-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-dihydroinden[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-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-2,3-dihydro-1H-inden-1,6-diol, 2-amino-6-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)-8H-inden[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)isoquinolin-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)phenyl)pyridin-2(1H)-one hydrochloride, 4-(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, 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)-1-methylpyridin-2(1H)-one, 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-((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)phenol, 5-(cyclopenta-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[1,2-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-(1-methyl-1H-imidazol-5-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]decane-2-yl)pyridazin-3-yl)-5-(1H-pyrazol-4-yl)phenol, and 4-(4-(6-(2,7-diazaspiro[4.5]decane-2-yl)pyridazin-3-yl)-3-hydroxyphenyl)-1-methylpyridin-2(1H)-one.,

[0078] Other representative splice modifiers have the following structures: TIFF2025521120000002.tif29128 with RG7916 (Roche / PTC / SMAF 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).

[0079] Other representative splice modifiers have the following structure: TIFF2025521120000003.tif46128 with RG7800 (Roche).

[0080] For example, the following are included: analogs of splicing modifiers such as RG7916 and RG7800 that can be used similarly: 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-hexahydropyrrolo[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-1-yl]pyrido[1,2-a]pyrimidin-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-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R,5S)-3,5-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)-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]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one, 7-fluoro-9-methyl-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-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof.,

[0081] IV. Method of Administration Administration or treatment can be carried out on any suitable cell or mammal by the methods or uses described herein. Typically, mammals having or suspected of having or expressing abnormal or non-normal proteins associated with a disease state are in need of the methods described herein.

[0082] Non-limiting examples of mammals include humans, non-human primates (apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), agricultural 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., 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., adult, teenager, child, infant or mammalian in utero). The mammal can be male or female. In certain embodiments, the mammal can be an animal disease model, such as, for example, an animal model having or expressing an abnormal or non-normal protein associated with a disease state, or an animal model in which protein expression is insufficient and that causes a disease.

[0083] Mammals (subjects) treated with the methods or compositions described herein include adults (18 years of age or older) and children (less than 18 years of age). Adults include the elderly. A representative adult is 50 years of age or older. The age of children ranges 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 include infants. Infants typically range from 1 - 12 months of age.

[0084] In certain embodiments, the method includes administering to a mammal a plurality of viral particles or nanoparticles, as described herein, to reduce, mitigate, prevent, inhibit, or delay the severity, frequency, progression, or time of onset of one or more symptoms of a disease state such as a neurodegenerative disease. In certain embodiments, the method includes administering to a mammal a plurality of viral particles or nanoparticles to treat a detrimental symptom of a disease state such as a neurodegenerative disease. In certain embodiments, the method includes administering to a mammal a plurality of viral particles or nanoparticles to stabilize, delay, or prevent the worsening, progression, or reversal of a disease state such as a neurodegenerative disease and its detrimental symptoms.

[0085] In certain embodiments, the method includes administering to a mammal a plurality of viral particles or nanoparticles to or into the central nervous system or a portion thereof, as described herein, to reduce, mitigate, prevent, inhibit, or delay the severity, frequency, progression, or time of onset of one or more symptoms of a disease state such as a neurodegenerative disease 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.

[0086] In certain embodiments, symptoms or detrimental effects include early, intermediate, or late symptoms, behavioral, personality, or speech symptoms, swallowing, movement, seizures, tremors, or restlessness symptoms, ataxia, and / or cognitive symptoms such as memory, the ability to organize.

[0087] In some embodiments, viral-based and non-viral-based gene transfer methods can be used to introduce nucleic acids into mammalian cells or mammalian target tissues. Such methods can be used to administer nucleic acids encoding inhibitory RNAs, therapeutic proteins, or components of the CRISPR system to cells in culture or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. Viral vector delivery systems include DNA viruses and RNA viruses that have an episomal genome or an integrated genome after delivery to the cell. To review 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.

[0088] Methods of non-viral delivery of nucleic acids include uptake of exosomes, lipofection, nucleofection, microinjection, biolistic methods, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced DNA. Lipofection is described in, for example, U.S. Patent 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 in WO 91117424, WO 91116024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or to a target tissue (e.g., in vivo administration).

[0089] In some embodiments, delivery is by use of a system for nucleic acid delivery based on an RNA virus or a DNA virus. In some aspects, the viral vector may be administered directly (in vivo) to a patient or used to treat cells in vitro or ex vivo and then administered to a patient. 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.

[0090] A. Viral Vector The term "vector" refers to a small carrier nucleic acid molecule, plasmid, virus (e.g., AAV vector, retroviral vector, lentiviral vector), or other vehicle that can be manipulated by insertion or incorporation of nucleic acid. Vectors such as viral vectors can be used to introduce / transfer nucleic acid into cells so that the nucleic acid sequence within the nucleic acid is transcribed by the cells and, if it encodes a protein, subsequently translated.

[0091] An "expression vector" is a special vector that contains a gene or nucleic acid sequence together with the essential regulatory regions necessary for expression in a host cell. An expression vector can contain at least an origin of replication for growth within the cell and optionally additional elements such as a heterologous nucleic acid sequence, expression control elements (e.g., promoter, enhancer), intron, ITR, and polyadenylation signal.

[0092] A viral vector is 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.

[0093] The term "recombinant" as a modifier of a vector, such as a recombinant viral vector, for example a recombinant lentivirus or a recombinant parvovirus (e.g., AAV) vector, and the term "recombinant" as a modifier of a sequence, such as a recombinant nucleic acid sequence or a recombinant polypeptide, means that the composition has been engineered (i.e., manipulated in a way that does not generally occur in nature). Specific examples of recombinant vectors such as AAV vectors, retroviral vectors or lentiviral vectors will have nucleic acid sequences inserted into the viral genome that are not normally present in the wild-type viral genome. An example of a recombinant nucleic acid sequence would be one that encodes an inhibitory RNA in which a nucleic acid (e.g., a gene) has been cloned into a vector with or without the 5' region, 3' region and / or intron regions that the gene is normally associated with 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.

[0094] 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 of the 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 has been 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 as compared to the viral genomic nucleic acid. Therefore, the incorporation of such non-native nucleic acid sequences defines that viral vector as a "recombinant" vector and, in the case of AAV, can be referred to as an "rAAV vector".

[0095] 1. Adeno-associated virus Adeno-associated virus (AAV) is a small non-pathogenic virus of the Parvoviridae family. To date, numerous serologically distinct AAVs have been identified, and more than 12 have been identified from humans or primates. AAV differs from other members of this family in that its replication depends on a helper virus.

[0096] The AAV genome exists episomally without integrating 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-level expression of the transduced gene. AAV virus particles are heat-stable, resistant to changes in solvents, detergents, pH, and temperature, and can be purified by column and / or concentrated by CsCl gradient or other means. The AAV genome contains single-stranded deoxyribonucleic acid (ssDNA) of either the plus or minus strand. The approximately 5 kb genome of AAV consists of one segment of single-stranded DNA with a plus or minus polarity. Both ends of the genome are short inverted terminal repeats (ITRs) that can fold into hairpin structures and function as origins of viral DNA replication.

[0097] The AAV "genome" ultimately refers to the recombinant nucleic acid sequence that is finally packaged or encapsulated to form AAV particles. 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 portions of the "plasmid" that do 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, processes necessary for propagation and production of recombinant virus, but which itself is not packaged or encapsulated into virus particles. Thus, the AAV vector "genome" refers to the nucleic acid packaged or encapsulated by AAV capsid proteins.

[0098] An AAV virion (particle) is a non-enveloped icosahedral particle approximately 25 nm in diameter. The AAV particle contains icosahedral symmetry composed of three related capsid proteins, VP1, VP2, and VP3, which interact with each other to form the capsid. The right ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins are often found in a ratio of 1:1:10, respectively, although the ratio may vary and all are derived from the right ORF. The VP1, VP2, and VP3 capsid proteins differ from each other by alternative splicing and use of an unusual start codon. Deletion analysis has shown that removal or modification of VP1 translated from an alternatively spliced message results in a reduction in the yield of infectious particles. Mutations within the VP3 coding region result in a failure to produce single-stranded progeny DNA or infectious particles.

[0099] An AAV particle is a virus particle containing an AAV capsid. In certain embodiments, the genome of the AAV particle encodes one, two, or all of the VP1, VP2, and VP3 polypeptides.

[0100] Most native AAV genomes often contain two open reading frames (ORFs), which may also be referred to as the left ORF and the right ORF. The left ORF often encodes the non-structural Rep proteins Rep40, Rep52, Rep68, and Rep78, which are involved in the regulation of replication and transcription in addition to the production of single-stranded progeny genomes. Two of the Rep proteins are associated with the preferential inclusion of the AAV genome into a region of the q arm of human chromosome 19. Rep68 / 78 has been shown to have NTP binding activity in addition to DNA helicase activity and RNA helicase activity. Some of the Rep proteins have nuclear localization signals along with several potential phosphorylation sites. In certain embodiments, the genome of AAV (e.g., rAAV) encodes some or all of the Rep proteins. In certain embodiments, the genome of AAV (e.g., rAAV) does not encode Rep proteins. In certain embodiments, one or more of the Rep proteins can be delivered in trans and thus are not included in AAV particles containing a nucleic acid encoding a polypeptide.

[0101] The ends of the AAV genome contain short inverted terminal repeats (ITRs) that have the potential to fold into a T-shaped hairpin structure that serves as the origin for viral DNA replication. Thus, the AAV genome contains one or more (e.g., a pair) of ITR sequences flanking the single-stranded viral DNA genome. The ITR sequences often each have a length of about 145 bases. Within the ITR region, two elements, the GAGC repeat motif and the terminal resolution site (trs), which are thought to be central to the function of the ITR, are described. 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, which occurs in a site- and strand-specific manner. These two elements appear to be central not only to their role in replication but also to viral encapsidation. The integration locus on chromosome 19 contains a Rep binding site adjacent to the trs. These elements have been shown to be functional and necessary for site-specific encapsidation.

[0102] In certain embodiments, AAV (e.g., rAAV) contains two ITRs. In certain embodiments, AAV (e.g., rAAV) contains a pair of ITRs. In certain embodiments, AAV (e.g., rAAV) contains a pair of ITRs flanking (i.e., present at the 5' and 3' ends of, respectively) a nucleic acid sequence encoding a polypeptide having at least function or activity.

[0103] An AAV vector (e.g., an rAAV vector) can be packaged and is referred to herein as an "AAV particle" for infection (transduction) of cells ex vivo, in vitro, or in vivo. When a recombinant AAV vector is encapsulated or packaged in an AAV particle, the particle can also be referred to as an "rAAV particle". In certain embodiments, the AAV particle is an rAAV particle. rAAV particles often contain an rAAV vector or a portion thereof. rAAV particles can be one or more rAAV particles (e.g., multiple AAV particles). rAAV particles typically contain proteins (e.g., capsid proteins) that encapsulate or package the rAAV vector genome. Note that reference to an rAAV vector can also be used to refer to an rAAV particle.

[0104] Any suitable AAV particle (e.g., an rAAV particle) can be used in the methods or uses herein. The rAAV particle and / or the genome contained therein can be derived from any suitable serotype or strain of AAV. The rAAV particle and / or the genome contained therein can be derived from two or more serotypes or strains of AAV. Thus, rAAV can contain proteins and / or nucleic acids or portions thereof of any serotype or strain of AAV, and the AAV particles are 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.

[0105] In certain embodiments, multiple rAAV particles contain 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, multiple rAAV particles contain a mixture of two or more different (e.g., different serotype and / or different strain) rAAV particles.

[0106] As used herein, the term "serotype" is a property used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological properties are determined based on the lack of cross-reactivity between antibodies to a particular AAV compared to another AAV. Such differences in cross-reactivity are usually due to differences in the capsid protein sequence / epitope (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of the AAV serotype). Even though AAV variants, including capsid variants, may not be serologically distinct from a reference AAV serotype or other AAV serotypes, they differ by at least one nucleotide residue or amino acid residue compared to the reference serotype or other AAV serotypes.

[0107] In certain embodiments, a particular serotype is excluded from the rAAV particles. In one embodiment, the rAAV particles are not AAV4 particles. In certain embodiments, the rAAV particles are antigenically or immunologically distinct from AAV4. The difference can be determined by standard methods. For example, ELISA and Western blot can be used to determine whether the viral particles are antigenically or immunologically distinct from AAV4. Additionally, in certain embodiments, the rAAV2 particles maintain a tissue tropism different from that of AAV4.

[0108] In certain embodiments, an rAAV vector based on a first serotype genome corresponds to one or more of the 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 particles.

[0109] In certain embodiments, the rAAV vector genome can be based on an AAV (e.g., AAV2) serotype genome derived from one or more of the AAV capsid proteins that package the vector. For example, the rAAV vector genome can contain nucleic acids (e.g., ITRs) derived 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 the AAV-2i8 serotype or variants thereof.

[0110] In certain embodiments, the rAAV particles or their vector genomes related to a reference serotype comprise or consist of a polynucleotide, polypeptide, or a subsequence thereof having 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 a subsequence thereof of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 particles. In certain embodiments, the rAAV particles or their vector genomes related to a reference serotype have 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 the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype.

[0111] In certain embodiments, the methods herein include the use, administration or delivery of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74 or rAAV-2i8 particles.

[0112] In certain embodiments, the methods herein include the use, administration or delivery of rAAV2 particles. In certain embodiments, the rAAV2 particles comprise an AAV2 capsid. In certain embodiments, the rAAV2 particles are at least 60%, 65%, 70%, 75% or more identical, 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% or up to 100% identical to the corresponding capsid proteins of native AAV2 particles or wild-type AAV2 particles, and include one or more capsid proteins (e.g., VP1, VP2 and / or VP3). In certain embodiments, the rAAV2 particles are at least 75% or more identical, such as 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% or up to 100% identical to the corresponding capsid proteins of native AAV2 particles or wild-type AAV2 particles, and include VP1, VP2 and VP3 capsid proteins. In certain embodiments, the rAAV2 particles are variants of native AAV2 particles 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 the capsid proteins of native AAV2 particles or wild-type AAV2 particles.

[0113] In certain embodiments, the rAAV9 particles comprise an AAV9 capsid. In certain embodiments, the rAAV9 particles are at least 60%, 65%, 70%, 75% or more identical, 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% or up to 100% identical, to the corresponding capsid proteins of native AAV9 particles or wild-type AAV9 particles, and comprise one or more capsid proteins (such as VP1, VP2 and / or VP3). In certain embodiments, the rAAV9 particles are at least 75% or more identical, such as 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% or up to 100% identical, to the corresponding capsid proteins of native AAV9 particles or wild-type AAV9 particles, and comprise VP1, VP2 and VP3 capsid proteins. In certain embodiments, the rAAV9 particles are variants of native AAV9 particles 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 the capsid proteins of native AAV9 particles or wild-type AAV9 particles.

[0114] In certain embodiments, the rAAV particles comprise one or two ITRs (e.g., a pair of ITRs) that are at least 75% identical, 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%, up to 100% 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, as long as they retain one or more desired ITR functions (e.g., the ability to form hairpins that allow DNA replication, the incorporation of AAV DNA into the host cell genome, and / or packaging if desired).

[0115] In certain embodiments, the rAAV2 particles comprise one or two ITRs (e.g., a pair of ITRs) that are at least 75% identical, 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%, up to 100% identical, to the corresponding ITRs of native or wild-type AAV2 particles, as long as they retain one or more desired ITR functions (e.g., the ability to form hairpins that allow DNA replication, the incorporation of AAV DNA into the host cell genome, and / or packaging if desired).

[0116] In certain embodiments, rAAV9 particles contain one or two ITRs (e.g., a pair of ITRs) that are at least 75% identical, 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%, up to 100% identical, to the corresponding ITRs of native or wild-type AAV2 particles, as long as they retain one or more desired ITR functions (e.g., the ability to form hairpins that enable DNA replication, the inclusion of AAV DNA into the host cell genome, and / or packaging if desired).

[0117] The rAAV particles can contain ITRs having any suitable number of "GAGC" repeats. In certain embodiments, the ITRs of AAV2 particles contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more "GAGC" repeats. In certain embodiments, the rAAV2 particles contain ITRs having 3 "GAGC" repeats. In certain embodiments, the rAAV2 particles contain ITRs having less than 4 "GAGC" repeats. In certain embodiments, the rAAV2 particles contain ITRs having more than 4 "GAGC" repeats. In certain embodiments, the ITRs of the rAAV2 particles contain a Rep binding site in which the fourth nucleotide in the first two "GAGC" repeats is C instead of T.

[0118] Examples of suitable lengths of DNA that can be incorporated into the rAAV vector for packaging / capsidation 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.

[0119] An rAAV vector containing a nucleic acid sequence that directs the expression of RNAi or a polypeptide can be produced using appropriate recombinant techniques known in the art (see, e.g., Sambrook et al., 1989). Recombinant AAV vectors are typically packaged into transducible AAV particles and propagated using an AAV virus packaging system. Transducible AAV particles have the ability to bind to and enter mammalian cells and subsequently deliver their nucleic acid cargo (e.g., a heterologous gene) to the nucleus of the cell. Thus, intact rAAV particles that are transducible are configured to transduce mammalian cells. rAAV particles configured to transduce mammalian cells are often non-replicable and require additional protein machinery to self-replicate. Thus, rAAV particles configured to transduce mammalian cells are engineered to bind to and enter mammalian cells and deliver nucleic acid to those cells, and the nucleic acid delivered is often positioned between a pair of AAV ITRs in the rAAV genome.

[0120] 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 recipients of heterologous rAAV vectors or that have been used as recipients of heterologous rAAV vectors. The stable human cell line HEK293 (readily available, for example, under accession number ATCC CRL1573 from the American Type Culture Collection) can be used. In certain embodiments, a modified human fetal kidney cell line (e.g., HEK293) that is transformed with an adenovirus type 5 DNA fragment and expresses the adenovirus E1a and E1b genes is used to produce recombinant AAV particles. The modified HEK293 cell line is readily transfected and provides a particularly convenient platform for producing rAAV particles. Methods for producing high-titer AAV particles having the ability to transduce mammalian cells are known in the art. For example, AAV particles can be made as described in Wright, 2008 and Wright, 2009.

[0121] In certain embodiments, the AAV helper function is introduced into host cells by transfecting the host cells with an AAV helper construct before or simultaneously with transfection of the AAV expression vector. Thus, in some cases, AAV helper constructs are used to at least transiently express the AAV rep gene and / or the cap gene for the purpose of complementing the 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 that encode both the Rep expression product and the Cap expression product. Several other vectors encoding the Rep expression product and / or the Cap expression product are also known.

[0122] 2. Retrovirus In the methods, compositions, and uses herein, viral vectors used as the agent to be delivered include retroviral vectors (see, e.g., Miller (1992) Nature, 357:455-460). Retroviral vectors are well-suited for delivery of nucleic acids to cells because they have the ability to deliver a single-copy gene that has not been rearranged to a wide range of rodent, primate, and human somatic cells. Retroviral vectors integrate into the genome of the host cell. Unlike other viral vectors, they infect only dividing cells.

[0123] Since retroviruses are RNA viruses, their viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate, which is very efficiently integrated into the chromosomal DNA of the infected cell. This integrated DNA intermediate is called a provirus. Transcription of the provirus and assembly into infectious virus occur either in the presence of a suitable helper virus or in cell lines containing appropriate sequences that allow encapsidation without the concomitant production of contaminating helper virus. If the sequences for encapsidation are provided by co-transfection of a suitable vector, the helper virus is not required for the production of recombinant retroviruses.

[0124] The retroviral genome and proviral DNA have three genes, gag, pol, and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes internal structural (matrix, capsid, and nucleocapsid) proteins, and the env gene encodes the viral envelope glycoproteins. The pol gene encodes products that include an RNA-directed DNA polymerase reverse transcriptase that transcribes the viral RNA into double-stranded DNA, an integrase that integrates the DNA produced by the reverse transcriptase into the host chromosomal DNA, and a protease that acts to process the encoded gag and pol genes. The 5' LTR and 3' LTR serve to facilitate transcription and polyadenylation of the virion RNA. The LTRs contain all other cis-acting sequences necessary for viral replication.

[0125] 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 can be either replication-competent or replication-defective. Typically, retroviral vectors are replication-defective, lacking the coding regions of genes required for new rounds of virion replication and packaging, or replaced with other genes. As a result, even if the virus infects an initial target cell, it cannot proceed along the typical cytolytic pathway. Such retroviral vectors and the agents (e.g., packaging cell lines) necessary to produce such viruses are commercially available (see, for example, retroviral vectors and retroviral systems such as catalog numbers 634401, 631503, 631501, etc. available from Clontech (Clontech, Mountain View, California)).

[0126] Such retroviral vectors can be produced as the agent to be delivered by replacing the viral genes necessary for replication with the nucleic acid molecule to be delivered. The resulting genome contains LTRs at both ends and one or more desired genes between them. Methods for producing retroviruses are known to those skilled in the art (see, for example, the published international PCT application WO1995 / 026411). Retroviral vectors can be produced in a packaging cell line containing one or more helper plasmids. The packaging cell line provides the viral proteins (such as the gag, pol, and env genes) necessary for vector capsid production and virion maturation. Typically, at least two separate helper plasmids (one containing the gag gene and the pol gene separately from the env gene) are used so that recombination between vector plasmids cannot occur. For example, retroviral vectors can be introduced into the packaging cell line using standard transfection methods such as calcium phosphate transfection. Packaging cell lines are well known to those skilled in the art and are commercially available. An exemplary packaging cell line is the GP2-293 packaging cell line (catalog numbers 631505, 631507, 631512, Clontech). After a 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 replication-incompetent recombinant retrovirus that contains the nucleic acid of interest but lacks the other structural genes so that new virus cannot be formed in the host cell.

[0127] As references illustrating the use of retroviral vectors in gene therapy, 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 may be cited.

[0128] 3. Lentivirus Lentivirus is a complex retrovirus that contains, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. Due to its high complexity, this virus can regulate its life cycle as seen in the course of latent infection. Examples of lentiviruses include human immunodeficiency virus: HIV-1, HIV-2, and simian immunodeficiency virus: SIV. Lentiviral vectors are produced by multiple attenuation of the HIV virulence genes. For example, by deleting the genes env, vif, vpr, vpu, and nef, the vector has become biologically safe. Lentiviral vectors are well known in the art (see, for example, U.S. Patent Nos. 6,013,516 and 5,994,136).

[0129] Recombinant lentiviral vectors have the ability to infect non-dividing cells and can be used for gene transfer and nucleic acid sequence expression both in vivo and ex vivo. For example, recombinant lentiviruses having the ability to infect non-dividing cells, in which appropriate host cells are transfected with two or more vectors responsible for packaging functions, namely gag, pol and env as well as rev and tat, are described in U.S. Patent No. 5,994,136 which is incorporated herein by reference.

[0130] The lentiviral genome and proviral DNA have three genes, gag, pol and env, found in retroviruses, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes internal structural (matrix, capsid and nucleocapsid) proteins, the pol gene encodes RNA-directed DNA polymerase (reverse transcriptase), protease and integrase, and the env gene encodes the viral envelope glycoprotein. The 5' LTR and 3' LTR serve to promote transcription and polyadenylation of virion RNA. The LTR contains all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx.

[0131] Adjacent to the 5' LTR are the sequences necessary for reverse transcription of the genome (tRNA primer binding site) and the sequences necessary for efficient encapsidation of viral RNA into particles (Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, cis defects prevent encapsidation of genomic RNA. However, the resulting mutants still retain the ability to direct the synthesis of all virion proteins.

[0132] 4. Other viral vectors The development and utility of viral vectors for gene delivery have been continuously improved and advanced. 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 can be selected according to the properties required for the target system.

[0133] 5. Chimeric viral vector Chimeric viral vectors or hybrid viral vectors have been developed for use in therapeutic gene delivery and their use in the present disclosure is contemplated. Chimeric provirus / retroviral vectors (Holzer et al., 1999), adenovirus / retroviral vectors (Feng et al., 1997, Bilbao et al., 1997, Caplen et al., 2000) and adenovirus / adeno-associated viral vectors (Fisher et al., 1996, U.S. Patent No. 5,871,982) have been described. In these "chimeric" viral gene transfer systems, the favorable characteristics of two or more parental virus species can be utilized. For example, Wilson et al. have provided a chimeric vector construct as described below that includes a portion of adenovirus, the 5' ITR sequence and 3' ITR sequence of AAV, and a selected transgene (U.S. Patent No. 5,871,983, which is hereby incorporated by reference in its entirety).

[0134] B. Nanoparticles 1. Lipid-based nanoparticles In some embodiments, the lipid-based nanoparticles are liposomes, exosomes, lipid formulations, or other lipid-based nanoparticles, such as lipid-based vesicles (e.g., DOTAP:cholesterol vesicles). The lipid-based nanoparticles can be positively charged, negatively charged, or neutral.

[0135] a. Liposomes "Liposomes" is a general term encompassing various unilamellar and multilamellar lipid vesicles formed by the generation of closed lipid bilayers or lipid aggregates. Liposomes can generally be characterized as having a vesicular structure with a bilayer membrane containing phospholipids and an inner medium generally containing an aqueous composition. The liposomes provided herein include unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. The liposomes provided herein can be positively charged, negatively charged, or neutrally charged. In certain embodiments, the liposomes are electrically neutral.

[0136] Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. Such liposomes are spontaneously formed when lipids containing phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-arrangement before the formation of a closed structure, 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.

[0137] In specific aspects, a polypeptide, nucleic acid, or small molecule drug can be encapsulated, for example, in the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule that associates with both the liposome and the polypeptide / nucleic acid, trapped within the liposome, or formed into a complex with the liposome.

[0138] The liposomes used according to this aspect can be made in a variety of ways known to those skilled in the art. For example, phospholipids, such as neutral phospholipid dioleoylphosphatidylcholine (DOPC), are dissolved in tert-butanol. Next, the lipid is mixed with polypeptides, nucleic acids, and / or other components. Tween 20 is added to this lipid mixture such that Tween 20 is about 5% by weight of the composition. An excess amount of tert-butanol is added to this 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 preparation is stored at -20°C and can be used for up to 3 months. If necessary, the lyophilized liposomes are reconstituted in 0.9% saline.

[0139] Alternatively, liposomes can be prepared by mixing lipids in a solvent in a container such as a glass pear-shaped flask. The container should have a volume 10 times larger than the volume of the expected liposome suspension. Using a rotary evaporator, the solvent is removed under reduced pressure at approximately 40°C. The solvent is usually removed within about 5 minutes to 2 hours, depending on the volume of the desired liposomes. The composition can be further dried in a desiccator under vacuum. The dried lipids tend to deteriorate over time and are generally discarded after about one week.

[0140] The dried lipids can be hydrated by shaking in sterile pyrogen-free water at about 25 - 50 mM phospholipid until the lipid film is resuspended. Next, the aqueous liposomes can be aliquoted, placed in vials, lyophilized, and sealed under vacuum.

[0141] The dried lipids or lyophilized liposomes prepared as described above are dehydrated and can be reconstituted in a solution of protein or peptide and diluted to an appropriate concentration with a suitable solvent, such as DPBS. Next, the mixture is vigorously stirred with a vortex mixer. Unencapsulated additional materials, such as, but not limited to, agents containing 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 material or additional active agent can be determined according to standard methods. After determining the amount of additional material or additional active agent encapsulated in the liposome preparation, the liposomes can be diluted to an appropriate concentration and stored at 4°C until use. A pharmaceutical composition containing liposomes will usually contain a sterile pharmaceutically acceptable carrier or diluent, such as water or saline solution.

[0142] As further liposomes that may be useful in this embodiment, cationic liposomes such as those described in WO02 / 100435A1, U.S. Patent No. 5,962,016, U.S. Patent Application Publication No. 2004 / 0208921, WO03 / 015757A1, WO04029213A2, U.S. Patent No. 5,030,453, and U.S. Patent No. 6,680,068 are mentioned, and these patent documents are hereby incorporated by reference in their entirety without exception into this specification.

[0143] When preparing such liposomes, any protocol described herein or any protocol 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, International Applications PCT / US85 / 01161 and PCT / US89 / 05040, each of which is hereby incorporated by reference into this specification.

[0144] In certain embodiments, the lipid-based nanoparticles are neutral liposomes (e.g., DOPC liposomes). As used herein, "neutral liposomes" or "uncharged liposomes" are defined as liposomes having one or more lipid components that impart an essentially neutral net charge (substantially uncharged). "Essentially neutral" or "essentially uncharged" means that, among the lipid components within a given population (e.g., a population of liposomes), if there are any that contain a charge not neutralized by the opposite charge of another component, they are present in only trace amounts (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 neutralized). In certain embodiments, the neutral liposomes can contain lipids and / or phospholipids that are themselves neutral under physiological conditions (i.e., at about pH 7).

[0145] The liposomes and / or lipid-based nanoparticles of this embodiment can contain phospholipids. In certain embodiments, one type of phospholipid can be used to produce the liposomes (e.g., a neutral phospholipid such as DOPC can be used to produce neutral liposomes). In other embodiments, two or more phospholipids can be used to produce the liposomes. The phospholipids can be derived from natural or synthetic sources. Examples of phospholipids include phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine. Phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., at about pH 7), and these compounds can be particularly useful for producing neutral liposomes. In certain embodiments, uncharged liposomes are produced using the phospholipid DOPC. In certain embodiments, lipids other than phospholipids (e.g., cholesterol) can be used.

[0146] Examples of phospholipids include glycerophospholipids and certain sphingolipids. Examples of phospholipids include dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauroylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoylphosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoylphosphatidylcholine ("SPPC"), dilauroylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), distearoylsphingomyelin ("DSSP"), distearoylphosphatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoylphosphatidic acid ("DMPA"), dipalmitoylphosphatidic acid ("DPPA"), dimyristoylphosphatidylethanolamine ("DMPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), dimyristoylphosphatidylserine ("DMPS"), dipalmitoylphosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoylsphingomyelin ("DPSP"), dimyristylphosphatidylcholine ("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine ("DBPC"), 1,2-docosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoyl phosphatidylethanolamine ("DOPE"), palmitoyloeoyl phosphatidylcholine ("POPC"), palmitoyloeoyl phosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine and dilinoleoyl phosphatidylcholine are included, but are not limited thereto.,

[0147] b. Exosome "Extracellular vesicles" and "EVs" are microvesicles derived from cells and secreted by cells, and include, as types, exosomes, exosome-like vesicles, ectosomes (produced by direct budding of vesicles from the plasma membrane), microparticles, microvesicles, shedding microvesicles (SMVs), nanoparticles, and also (large) apoptotic blebs or apoptotic bodies (produced by cell death) or membrane particles.

[0148] As used herein, the terms "microvesicle" and "exosome" refer to membrane particles having a diameter (or maximum dimension if the particle is not 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, and at least a portion of the membrane of the exosome is directly obtained from the cell. Most commonly, exosomes will have a size (average diameter) up to 5% of the size of the donor cell. Therefore, particularly contemplated exosomes include those shed from cells.

[0149] Exosomes can be detected in or isolated from any suitable sample type, such as body 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 can be any sample containing exosomes suitable for detection or isolation. Sources of the sample include blood, bone marrow, pleural effusion, ascites, 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, for example, whole blood or any fraction or component thereof. A blood sample suitable for use in the present invention can be extracted from any known source containing blood cells or their components, such as venous blood, arterial blood, peripheral blood, tissue blood, umbilical cord blood, etc. For example, the sample 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.

[0150] Exosomes can also be isolated from tissue samples, such as surgical samples, biopsy samples, tissues, feces, and cultured cells. When isolating exosomes from a tissue source, it will be necessary to homogenize the tissue to obtain a single cell suspension and then lyse those cells to release the exosomes. When isolating exosomes from a tissue sample, it is important to select homogenization and lysis techniques that do not result in fragmentation of the exosomes. The exosomes contemplated herein are preferably isolated from body fluids in a physiologically acceptable solution, such as buffered saline, growth medium, various aqueous media, etc.

[0151] Exosomes can be isolated from freshly collected samples or from samples that have been stored frozen or refrigerated. In some embodiments, exosomes can be isolated from cell culture media. Although not necessary, if the liquid sample is clarified prior to precipitation with a volume-excluding polymer to remove any debris from the sample, higher purity exosomes will likely be obtained. Methods of clarification include centrifugation, ultracentrifugation, filtration, or tangential flow filtration. Most typically, exosomes can be isolated by numerous 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).

[0152] One generally recognized exosome isolation protocol 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 sequential differential centrifugation is complicated by the potential for size distribution overlap with other microvesicles or macromolecular complexes. Furthermore, centrifugation may be insufficient as a means of separating vesicles based on their size. However, combining sequential centrifugation with sucrose gradient ultracentrifugation can result in high enrichment of exosomes.

[0153] Size-based isolation of exosomes using methods alternative to ultracentrifugation is another option. Successful purification of exosomes using ultrafiltration techniques has been reported, which takes less time than ultracentrifugation and does not require special equipment. Similarly, commercially available kits (EXOMIR™, Bioo Scientific) can be used that enable the removal of cells, platelets, and cell debris with one microfilter and the capture of vesicles larger than 30 nm with a second microfilter by driving fluid using positive pressure. However, in this process, exosomes are not recovered, and their RNA content is directly extracted from the material captured on the second microfilter and can be used for PCR analysis. HPLC-based protocols may potentially enable the obtainment of highly pure exosomes, but these processes require dedicated equipment and are difficult to scale up. A major problem is that both blood and cell culture media contain numerous nanoparticles (some non-vesicular) in the same size range as exosomes. For example, some miRNAs can be contained within extracellular protein complexes other than exosomes, but treatment with proteases (e.g., proteinase K) can remove possible contamination with "extra-exosomal" proteins.

[0154] In another aspect, exosomes can be captured by techniques commonly used to concentrate samples for exosomes, such as techniques involving immunospecific interactions (e.g., immunomagnetic capture). Immunomagnetic capture is also known as immunomagnetic cell separation and typically requires attaching an antibody to a small paramagnetic bead that binds to a protein found on a specific cell type. When the antibody-coated beads are mixed with a sample such as blood, they bind to and surround those specific cells. Next, the sample is placed in a strong magnetic field to pellet the beads in one direction. Even after removal of the blood, the captured cells are retained with the beads. Numerous variations of this general method are well 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 an antibody is added to the mixture to recognize epitopes on the surface of the exosomes bound to the beads.

[0155] As will be understood by those skilled in the art, exosomes may be further modified by including targeting moieties, either before or after loading the cargo, for the purpose of enhancing their utility as vehicles for delivering the cargo. In this regard, exosomes may be engineered to incorporate entities that specifically target a particular cell type or tissue type. Such target-specific entities, such as peptides having an affinity for receptors or ligands on the target cell or target tissue, can be incorporated into the membrane of the exosome, for example, by fusion to exosome membrane markers using methods established in the art.

[0156] 2. Non-lipid nanoparticles Spherical Nucleic Acid (SNA (trademark)) constructs and other nanoparticles, particularly gold nanoparticles, are also contemplated as means for delivering chimeric mini-genes to intended target cells. Because of their dense 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 exhibit transfection efficiencies of 99% without the need for transfection agents or transfection reagents. The unique target-binding affinity and specificity of these constructs allow for excellent specificity for the matched target sequence (i.e., limited off-target effects). These constructs are significantly superior to major conventional transfection reagents (Lipofectamine 2000 and Cytofectin). These constructs can enter a variety of cultured cells, primary cells, and tissues without obvious toxicity. The changes in global gene expression measured by whole-genome microarray studies and cytokine-specific protein assays induced by these constructs are minimal. Any number of single or combined agents (e.g., proteins, peptides, small molecules) can be used for tailoring the surface of these constructs. See, for example, Jensen et al., Sci. Transl. Med. 5, 209ra152 (2013).

[0157] Self - assembling nanoparticles with nucleic acid cargo can be constructed by using PEGylated polyethyleneimine (PEI) and attaching an Arg - Gly - Asp (RGD) peptide ligand to the distal end of polyethylene glycol (PEG). Nanoplexes can be prepared by mixing equal volumes of an aqueous solution of a cationic polymer and an aqueous solution of nucleic acid, such that the net molar excess of ionizable nitrogen (polymer) over phosphate (nucleic acid) ranges from 2 to 6. Electrostatic interactions between the cationic polymer and the nucleic acid result in the formation of polyplexes. Since this has an average particle size distribution of about 100 nm, it is called a nanoplex (see, for example, Bartlett et al., PNAS, 104:39, 2007).

[0158] C. Encapsulated cell transplantation The chimeric mini - genes herein can be delivered to cells ex vivo, and then it is encapsulated and transplanted to deliver the target gene to the patient. For example, cells isolated from a patient or a donor into which exogenous heterologous nucleic acid has been introduced can be directly delivered to the patient by transplantation of encapsulated cells. The advantage of transplanting encapsulated cells is that the immune response against the cells is reduced by encapsulation. Thus, herein, a method for symmetric administration of genetically modified cells is provided. The number of cells to be delivered depends on the desired effect, the specific nucleic acid, the subject to be treated, and other similar factors, and those skilled in the art can determine it.

[0159] Cells into which nucleic acids are introduced for gene therapy include any desirable available cell type, such as 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 cells 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, but are not limited thereto. For example, the genetically modified cells can be pluripotent stem cells or totipotent stem cells (including induced pluripotent stem cells), or 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 a different species as the recipient subject. In one preferred example, the cells used in gene therapy are autologous cells for the patient. Methods for genetically modifying cells and methods for transplanting cells are known in the art.

[0160] Typically, first a nucleic acid is introduced into a cell, and then the resulting recombinant cell is administered in vivo. Such introduction can be effected by any method known in the art, for example, but not limited to, transfection, electroporation, microinjection, infection with a viral vector or bacteriophage vector containing the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like. 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, Cotton et al., Meth. Enzymol. (1993) 217:618-644, Cline, Pharmac. Ther. (1985) 29:69-92), and these can be used so long as they do not disrupt the necessary developmental and physiological functions of the recipient cell. In certain instances, the method enables stable transfer of the nucleic acid into the cell such that the nucleic acid can be expressed by the cell and can also be inherited by and expressed by its progeny cells.

[0161] Encapsulation can be carried out using alginate microcapsules coated with an alginate / polylysine complex. Hydrogel microcapsules have been widely studied for the encapsulation of living cells or aggregates of living cells 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). Generally, capsules are designed to release therapeutic proteins secreted by cells, allow easy diffusion of oxygen and nutrients to the encapsulated cells, and protect the cells from attack by the immune system. They have been developed as potential therapeutic agents for a range of diseases including neurodegenerative disorders such as type I diabetes, cancer and 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 cross-linking. In a typical process, first, a viscous alginate solution is formulated with the cells. Next, the cell suspension is processed into microdroplets using various methods such as air shear, acoustic vibration or electrostatic droplet formation (Rabanel et al. Biotechnol. Prog. 2009, 25, 946). Alginate droplets gel upon contact with a solution of divalent ions such as Ca2+ or Ba2+.

[0162] A capsule for transplanting mammalian cells into a subject is disclosed. The capsule is formed from a biocompatible hydrogel-forming polymer that encapsulates the cells to be transplanted. To inhibit capsular overgrowth (fibrosis), the structure of the capsule prevents cellular material from locating on the capsule surface. In addition, the structure of the capsule 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 encapsulated therein for controlled release.

[0163] The disclosed composition is formed from a biocompatible hydrogel-forming polymer that encapsulates the cells to be transplanted. Examples of materials that can be used to form a suitable hydrogel include polysaccharides such as alginate, collagen, chitosan, sodium cellulose sulfate, gelatin, and agarose, water-soluble polyacrylates, polyphosphazines, poly(acrylic acid), poly(methacrylic acid), poly(alkylene oxide), poly(vinyl acetate), polyvinylpyrrolidone (PVP), and their copolymers and blends. See, for example, U.S. Patent Nos. 5,709,854, 6,129,761, 6,858,229, and 9,555,007.

[0164] V. Pharmaceutical Compositions As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" mean 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" composition or a "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material can be administered to a subject without substantially causing undesirable biological effects. Such compositions, "pharmaceutically acceptable" and "physiologically acceptable" formulations and compositions can be sterile. Such pharmaceutical formulations and pharmaceutical compositions can be used, for example, when administering viral particles or nanoparticles to a subject.

[0165] 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 media and suspending media, coatings, isotonicity and absorption promoting or absorption delaying agents, which are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions can include suspending and thickening agents. Auxiliary active compounds (e.g., preservatives, antibacterial agents, antiviral agents and antifungal agents) can also be incorporated into the formulations and compositions.

[0166] Pharmaceutical compositions typically contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of harmful antibodies in the individual to whom the composition is administered and can be administered without undue toxicity. Examples of pharmaceutically acceptable excipients include, but are not limited to, liquids such as sorbitol, Tween80, water, saline, glycerol and ethanol. It can include pharmaceutically acceptable salts, such as mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and salts of organic acids such as acetate, propionate, malonate, benzoate. Furthermore, auxiliary substances, such as surfactants, wetting or emulsifying agents, pH buffering substances, etc., can also be present in such vehicles.

[0167] The pharmaceutical composition can be formulated to be compatible with a particular route of administration or delivery shown herein or known to those skilled in the art. Accordingly, the pharmaceutical composition includes carriers, diluents, or excipients suitable for administration or delivery by various routes.

[0168] Pharmaceutical forms suitable for injection of virus particles or nanoparticles are adapted for the immediate preparation of sterile injectable or infusible solutions or dispersions, and can include sterile aqueous solutions or aqueous dispersions, optionally encapsulated in liposomes. In either case, the final form should be a 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. Appropriate fluidity can be maintained, for example, by the formation of liposomes, by maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Isotonic agents, such as sugars, buffers, or salts (such as sodium chloride) can be included. Sustained absorption of the injectable composition can be brought about by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0169] A solution or suspension of virus particles or nanoparticles can optionally contain one or more of the following components: a sterile diluent, such as water for injection, a saline solution, such as phosphate buffered saline (PBS), artificial CSF, a surfactant, a fixed oil, a polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), glycerin, or other synthetic solvents, antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, etc.; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate, and an agent for adjusting tonicity, such as sodium chloride or dextrose.

[0170] Pharmaceutical formulations, compositions, and delivery systems suitable for the compositions, methods, and uses of the present invention are known in the art (e.g., Remington: The Science and Practice of Pharmacy (2003) 20 th ed., Mack Publishing Co., Easton, Pennsylvania, Remington's Pharmaceutical Sciences (1990) 18 th ed., Mack Publishing Co., Easton, Pennsylvania, The Merck Index (1996) 12 th ed., Merck Publishing Group, Whitehouse, New Jersey, Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pennsylvania, Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11 thSee, e.g., ed., Lippincott Williams & Wilkins, Baltimore, Maryland, and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, New York, pp. 253-315).

[0171] Viral particles, nanoparticles, and their compositions can be formulated into dosage unit forms to facilitate administration and provide uniform dosing. As used herein, "dosage unit form" refers to physically discrete units suitable as unit dosages for the individual to be treated, each unit containing a predetermined quantity of the active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The dosage unit form depends on the number of viral particles or nanoparticles considered necessary to produce the desired effect. The required amount can be formulated as a single dose or in multiple dosage units. The dosage can be adjusted to an appropriate viral particle concentration or nanoparticle concentration, optionally combined with an anti-inflammatory agent, and packaged for use.

[0172] In one aspect, the pharmaceutical composition will contain a therapeutically effective amount of a genetic material, i.e., an amount sufficient to reduce or ameliorate the symptoms or adverse effects of the disease state in question, or to provide a desired benefit.

[0173] As used herein, the term "unit dosage form" refers to a physically discrete unit suitable as a unit dose for administration to a subject to be treated, each unit containing a predetermined quantity calculated to produce the desired effect (e.g., a prophylactic or therapeutic effect) when administered one or more times, optionally together with a pharmaceutical carrier (excipient, diluent, vehicle or filler). The unit dosage form may be, for example, in ampoules and vials containing a liquid composition, a composition in a lyophilized or freeze-dried state, and a sterile liquid carrier may be added thereto, for example, prior to in vivo administration or in vivo delivery. Individual unit dosage forms can be included in a multi-dose kit or container. Thus, for example, virus particles, nanoparticles and their pharmaceutical compositions can be packaged in single or multiple unit dosage forms to facilitate administration and provide uniform dosing.

[0174] Formulations containing virus particles or nanoparticles typically contain an effective amount, which can be readily determined by one of ordinary skill in the art. Virus particles or nanoparticles can typically be in the range of about 1% to about 95% (w / w) of the composition, or higher if appropriate. The amount administered depends on factors such as the age, weight and health status of the mammalian or human subject being treated. One of ordinary skill in the art can establish the effective dosage by routine testing to establish a dose-response curve.

[0175] VI. Definitions 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 their polymers. Polynucleotides include genomic DNA, cDNA and antisense DNA, as well as spliced or unspliced mRNA, rRNA, tRNA and inhibitory DNA or inhibitory RNA (RNAi, e.g., small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA or antisense RNA). Polynucleotides can include natural, synthetic, and intentionally modified or engineered polynucleotides (e.g., variant nucleic acids). Polynucleotides can be single-stranded, double-stranded or triple-stranded, linear or circular, and can be of any suitable length. In discussions of polynucleotides, the sequence or structure of a particular polynucleotide can be described herein according to the convention of describing sequences in the 5'→3' direction.

[0176] Nucleic acids encoding polypeptides often contain an open reading frame encoding the polypeptide. Unless otherwise indicated, a particular nucleic acid sequence also includes degenerate codon substitutions.

[0177] A nucleic acid can include one or more expression control or regulatory elements operably linked to an open reading frame, and the one or more regulatory elements are configured to direct transcription and translation of the polypeptide encoded by the open reading frame in mammalian cells. 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.

[0178] "Promoter" usually refers to a nucleotide sequence that is located upstream (5'-side) of the coding sequence and directs and / or controls the expression of the coding sequence by providing recognition sites for RNA polymerase and other factors necessary for proper transcription. A "promoter" includes a minimal promoter, which is a short DNA sequence composed of a TATA box and, optionally, other sequences that function to specify the transcription start site, to which regulatory elements for controlling expression are added.

[0179] An "enhancer" is a DNA sequence that can stimulate transcriptional activity and can be an element specific to the promoter or a heterologous element that enhances the level of expression or the tissue specificity of expression. It can operate in either direction (5'→3' or 3'→5') and has the ability to function regardless of whether it is located upstream or downstream of the promoter.

[0180] A promoter and / or enhancer may be entirely derived from the 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 a DNA sequence involved in the binding of protein factors that regulate / control the effectiveness of transcription initiation in response to stimuli, physiological conditions, or developmental conditions.

[0181] As non-limiting examples, there may be mentioned the SV40 early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the pol II promoter, the pol III promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from non-viral genes, such as the mouse metallothionein gene, may also be useful here. Exemplary constitutive promoters include the promoters of the following genes encoding a specific constitutive function or "housekeeping" function, and other constitutive promoters known to those skilled in the art: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, the actin promoter. In addition, many viral promoters function constitutively in eukaryotic cells. These include, inter alia, 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. Thus, any of the above-described constitutive promoters can be used to control the transcription of a heterologous gene insert.

[0182] As used herein, for convenience, a nucleic acid sequence / polynucleotide introduced into or introduced into a cell or organism is referred to as a "transgene". Transgenes include any nucleic acid, such as a gene encoding an inhibitory RNA or a polypeptide or protein, which are generally heterologous to the native AAV genomic sequence.

[0183] 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. When the introduced transgene is integrated into the nucleic acid (genomic DNA) of the recipient cell or recipient organism, it can be stably maintained in that cell or organism and further transmitted or inherited by the progeny cells or progeny organisms of the recipient cell or recipient organism. Finally, the introduced transgene may exist extrachromosomally or only transiently in the recipient cell or recipient host organism. Therefore, 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 its progeny. Transduced cells can be propagated, the transgene can be transcribed, and the encoded inhibitory RNA or protein can be expressed. In the case of use in gene therapy and methods of gene therapy, transduced cells can be present in a mammal.

[0184] 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 an inducer (for example, transcription under the control of the metallothionein promoter is significantly increased in the presence of certain metal ions). Inducible promoters contain responsive elements (REs) that stimulate transcription when their respective inducing factors bind. For example, there are REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. A promoter containing a specific RE can be chosen to obtain an inducible response, and in some cases, the RE itself can be ligated to a different promoter to confer inducibility on the recombinant gene. Therefore, by selecting an appropriate promoter (constitutive or inducible, strong or weak), it is possible to control both the presence and the expression level of a polypeptide in genetically modified cells. When a gene encoding a polypeptide is under the control of an inducible promoter, in situ delivery of the polypeptide is triggered by exposing the in situ genetically modified cells to conditions that permit transcription of the polypeptide, for example, by intraperitoneal injection of a specific inducer of the inducible promoter that controls transcription of the agent. For example, in situ expression of a polypeptide encoded by a gene under the control of the metallothionein promoter is enhanced by contacting the genetically modified cells in situ with a solution containing the appropriate (i.e., inducible) metal ions.

[0185] A nucleic acid / transgene is said to be "functionally linked" when it is placed in a functional relationship with another nucleic acid sequence. A nucleic acid / transgene encoding RNAi or a polypeptide or a nucleic acid directing the expression of a polypeptide may contain an inducible or tissue-specific promoter to control transcription of the encoded polypeptide. A nucleic acid functionally linked to an expression control element can also be called an expression cassette.

[0186] In certain embodiments, in the methods and uses described herein, CNS-specific or inducible promoters, enhancers, etc. are used. 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 response elements for ecdysone, tetracycline, hypoxia, and IFN.

[0187] 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.

[0188] As used herein, the terms "modify" or "variant" and their grammatical variants mean that a nucleic acid, polypeptide, or a subsequence thereof deviates from a reference sequence. Therefore, the modified sequence and the variant sequence may have an expression level, activity, or function that is substantially the same as, greater than, or less than that of the reference sequence, but at least partially retains the activity or function of the reference sequence. A particular type of variant is a mutant protein, which refers to a protein encoded by a gene having a mutation such as a missense mutation or a nonsense mutation.

[0189] A "nucleic acid" variant or "polynucleotide" variant refers to a modified sequence in which the gene is changed as compared to the wild type. The sequence can be genetically modified without changing the encoded protein sequence. Alternatively, the sequence can be genetically modified to encode a variant protein. A nucleic acid variant or polynucleotide variant can also refer to a combinatorial sequence that is codon-modified 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-modified to encode a variant protein. For example, some codons of such a nucleic acid variant change so as not to change the amino acid of the protein encoded thereby, and some codons of the nucleic acid variant change such that as a result, it will change the amino acid of the protein it encodes.

[0190] The terms "protein" and "polypeptide" are used interchangeably herein. The "polypeptides" encoded by the "nucleic acids" or "polynucleotides" or "transgenes" disclosed herein include partial-length or full-length native sequences, as well as native wild-type and functional polymorphic proteins, functional partial sequences (fragments) thereof, and sequence variants thereof, as long as the polypeptide retains some function or activity. Thus, in the methods and uses of the present invention, such polypeptides encoded by a nucleic acid sequence need not be identical to an endogenous protein that is defective, or whose activity, function or expression is insufficient, lacking or absent in the mammal being treated.

[0191] Non-limiting examples of modifications include substitutions 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).

[0192] Examples of amino acid modifications are conservative amino acid substitutions or deletions. In certain embodiments, the modified sequence or variant sequence retains at least a portion of the function or activity of the unmodified sequence (e.g., wild-type sequence).

[0193] Another example of an amino acid modification is a targeting peptide introduced into the capsid protein of a viral particle. Peptides that target recombinant viral vectors or nanoparticles to the central nervous system, e.g., vascular endothelial cells, have been identified. Thus, modified recombinant viral particles or nanoparticles can target, for example, endothelial cells lining the cerebral blood vessels.

[0194] A recombinant virus so modified can bind preferentially to one type of tissue (e.g., CNS tissue) over another type of tissue (e.g., liver tissue). In certain embodiments, a recombinant virus retaining a modified capsid protein can "target" cerebrovascular epithelial tissue by binding at a higher level than a comparable unmodified capsid protein. For example, a recombinant virus having a modified capsid protein can bind to cerebrovascular epithelial tissue at a level 50% to 100% higher than an unmodified recombinant virus.

[0195] 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 involved in the transfer of information contained within DNA to proteins. Fragments and variants of the disclosed nucleotide sequences, as well as the proteins or partial-length proteins encoded thereby, are also encompassed by the present invention. A "fragment" or "portion" means a nucleotide sequence or amino acid sequence of a polypeptide or protein that encodes a full-length or less-than-full-length 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).

[0196] A "variant" of a molecule is an array that is substantially similar to the sequence of the native molecule. In the case of a nucleotide sequence, variants include sequences that encode 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 techniques of molecular biology such as, for example, polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include nucleotide sequences of synthetic origin, such as those prepared using site-directed mutagenesis that encode the native protein, as well as those that encode polypeptides having amino acid substitutions. In general, the nucleotide sequence variants of the present invention have at least 40%, 50%, 60%, or 70%, for example 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, generally at least 80%, for example 81% - 84%, at least 85%, for example 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 variant 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).

[0197] "Conservative substitution" of a particular nucleic acid sequence refers to a nucleic acid sequence that encodes an amino acid sequence that is identical or essentially identical. Because the genetic code is degenerate, any given polypeptide is encoded by a number of functionally identical nucleic acids. For example, the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, anywhere an arginine is specified by a codon, the codon can be changed to any of the described corresponding codons without changing the encoded protein. Such nucleic acid variations are "silent variations" and are a type of "conservatively modified variation." Each of the nucleic acid sequences described herein that encode a polypeptide represents all possible silent variations, unless otherwise noted. It will be appreciated by those skilled in the art that each codon in a nucleic acid (except for the 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 encoding a polypeptide is implicitly represented by each of the sequences described.

[0198] The term "substantial identity" of a polynucleotide sequence means that the polynucleotide, when compared to a reference sequence using one of the described alignment programs with standard parameters, has 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. It will be understood by those skilled in the art that these values can be appropriately adjusted considering codon degeneracy, amino acid similarity, reading frame position, etc. when determining the corresponding identity of the proteins encoded by two nucleotide sequences. For these purposes, substantial identity of an amino acid sequence generally means at least 70%, at least 80%, 90%, or even at least 95% sequence identity.

[0199] The term "substantial identity" with respect to a polypeptide indicates that the polypeptide, in a specified comparison window, has 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. A measure that two polypeptide sequences are identical is that one polypeptide immunoreacts with an antibody raised against the other polypeptide. Thus, for example, if the differences between two polypeptides are only conservative substitutions, the polypeptide is identical to the other polypeptide.

[0200] The terms "treating" and "treatment" refer to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent, inhibit, reduce or decrease an undesirable physiological change or physiological disorder, e.g., the occurrence, progression or exacerbation of a disorder. In the context of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, diminution of the extent of a disease, stabilization of a disease symptom or adverse effect (i.e., no exacerbation or progression), delay or deceleration of disease progression, improvement or alleviation of a disease state, and remission (whether partial or complete). "Treatment" can also mean an extension of survival as compared to the expected survival in the absence of treatment. Those in need of treatment include those already having a condition or disorder, as well as those having a predisposition (e.g., as determined by a genetic assay).

[0201] The terms "comprising", "having", "including" and "containing" should be construed as being non-limiting terms (i.e., meaning "including but not limited to") unless otherwise noted.

[0202] All methods and uses described herein can be performed in any suitable order, unless otherwise indicated herein or unless clearly inconsistent with the context. The use of any and all examples or exemplary language (e.g., "such as" or "for example") provided herein is for illustrative purposes only and is not intended to limit the scope of the invention unless otherwise claimed. No representation herein should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0203] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by alternative features that serve the same purpose, equivalent purpose or similar purpose. Thus, unless otherwise explicitly stated, the disclosed features (e.g., modified nucleic acids, vectors, plasmids, recombinant vector sequences, vector genomes, or virus particles) are an example of a genus of equivalent or similar features.

[0204] As used herein, the forms “a”, “and” and “the” include singular and plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of such nucleic acids, reference to “a vector” includes a plurality of such vectors, and reference to “a virus” or “AAV or rAAV particle” includes a plurality of such virions / AAV or rAAV particles.

[0205] As used herein, “essentially free of” with respect to a specified component is used to mean that in this specification, the specified component is not intentionally formulated in the composition and / or is present only as an impurity or in trace amounts. Therefore, the total amount of the specified component due to any unintentional contamination of the composition is far lower than 0.05%, preferably less than 0.01%. Most preferably, the composition is one in which the amount of the specified component cannot be detected by standard analytical methods.

[0206] As used in this specification, “a” or “an” can mean one or more. As used in the claims of this application, the words “a” or “an” used in conjunction with the word “comprising” can mean one or a number greater than one.

[0207] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to only alternative options or the alternative options are mutually exclusive. However, this disclosure supports both a definition that refers to only alternative options and a definition that refers to "and / or". As used herein, "another" can mean at least one more, or more than one.

[0208] Throughout this application, the term "about" is used to indicate that a value includes a value that is within 10% of the stated value, the variation in error inherent in the apparatus, method used to determine that value, the variation that exists between study subjects, or both.

[0209] The recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein.

[0210] Accordingly, all numerical values or numerical ranges include integers within such ranges and fractions of integers within such values or ranges, unless the context clearly indicates otherwise. Thus, for example, "80% or more 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., and so on.

[0211] When referring to an integer with the words "more (greater) or less", it includes all numbers greater than or less than that reference number. Thus, for example, when saying less than 100, it includes all numbers from 99, 98, 97, etc. down to the number 1, and when saying less than 10, it includes all numbers from 9, 8, 7, etc. down to the number 1.

[0212] As used herein, all numerical values or numerical ranges include the fractional parts of those values, as well as the integers within such ranges and the fractional parts of the integers within such ranges, unless the context clearly indicates otherwise. Thus, for example, a reference to a numerical range such as 1 to 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. Therefore, a reference to the range of 1 to 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.

[0213] A reference to a series of ranges includes the ranges formed by combining the boundary values of the different ranges therein. Thus, for example, a reference to a series of ranges such as 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 75, 75 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 400, 400 to 500, 500 to 750, 750 to 1,000, 1,000 to 1,500, 1,500 to 2,000, 2,000 to 2,500, 2,500 to 3,000, 3,000 to 3,500, 3,500 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, etc. includes 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.

[0214] VII. Kit The present invention provides a kit with a packaging material and one or more components therein. The kit typically includes a label or an accompanying document that contains a description of the components included therein or instructions regarding the in vitro, in vivo, or ex vivo use of the components included therein. The kit can include 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.

[0215] A kit refers to a physical structure that houses one or more components of the kit. The packaging material can be made of materials commonly used for such purposes (e.g., paper, cardboard, glass, plastic, foil, ampoules, vials, tubes, etc.) that can maintain the components aseptically.

[0216] The label or the accompanying document can include identification information of one or more components included therein, dosage, clinical pharmacology of the active ingredients, such as mechanism of action, pharmacokinetics, and pharmacodynamics. The label or the accompanying document can include information for verifying the manufacturer, lot number, manufacturing location, and manufacturing date, and expiration date. The label or the accompanying document can include information regarding the diseases for which the components of the kit can be used. The label or the accompanying document can include instructions for clinicians or subjects for using one or more of the components of the kit in a method, use, or treatment protocol or therapeutic regimen. The instructions can include dosage, dosing frequency or duration, and instructions for implementing any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0217] The label or package insert can include information regarding the benefits that the composition can provide, such as prophylactic or therapeutic benefits. The label or package insert can include information regarding potentially harmful side effects, complications, or reactions, such as warnings to the subject or clinician regarding situations in which it may not be appropriate to use a particular composition. Harmful side effects or complications can occur if the subject has taken or will take or is taking one or more other medicaments that may be incompatible with the composition, or if the subject has undergone or will undergo or is undergoing another treatment protocol or therapeutic regimen that may be incompatible with the composition, and thus the instructions can also include information regarding such incompatibilities.

[0218] The label or package insert includes "printed matter", such as paper or cardboard, that is separate or attached to the composition, kit, or packaging material (e.g., box), or attached to an ampoule, tube, or vial containing a component of the kit. The label or package insert can further include a computer-readable medium, such as a printed label with a barcode, disk, optical disk, such as a CD- or DVD-ROM / RAM, DVD, MP3, or electronic storage media, such as RAM and ROM, or hybrids thereof, such as magnetic / optical storage media, flash memory, hybrid, and memory type cards.

Examples

[0219] VIII. Examples The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should recognize that the techniques disclosed in the following examples represent techniques discovered by the inventor to function well in the practice of the invention and, thus, can constitute preferred modes for its practice. However, those skilled in the art should recognize that, in light of the present disclosure, many modifications can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0220] Example 1-Regulatory control of gene therapy by a drug-inducible switch We previously developed a switch-on cassette from an LMI070-responsive exon, including that from the minimal intron-spanning sequence required to reiterate splicing of a pseudoexon in SF3B3 (SF3B3.X on ;X on ). Placing these earlier cassettes upstream of SaCas9 resulted in a complete cassette that exceeded the current AAV packaging limit (5.6 kb). Therefore, we generated a smaller version, designated SF3B3.miniX on (miniX on ;miX on ) (Figure 1A). Induction and splicing of miniX on were maintained, and its overall amplitude was only slightly reduced compared to SF3B3-X on ; at 100 nM LMI070, induction of miniX on was 132-fold compared to 152-fold of X on (Figures 1B-1C). In addition to AAV8-miniX on -SaCas9, a single-guide RNA targeting the loxP-STOP sequence was intravenously administered to Ai14 mice; after several weeks, LMI070 was administered and gene editing was evaluated 7 days later (Figure 1D). Expression of tdTomato was detected by fluorescence-activated cell sorting analysis of hepatocytes and histology of liver tissue sections (Figures 1E-1F). Furthermore, gene editing was confirmed by genomic DNA PCR assay followed by Sanger sequencing (Figures 1G-1H). Cumulatively, these results expanded the utility of X on to control translation of Cas9 protein for gene editing applications.

[0221] The sequences of the SF3B3.X on cassette and the miniX on cassette are as follows.

[0222] SF3B3.X on : TIFF2025521120000004.tif98146

[0223] SF3B3.miniX on : TIFF2025521120000005.tif49146

[0224] SF3B3.X on and SF3B3.miniX on Provide further breakdowns of the arrays that make up and SF3B3.miniX in Tables 1 and 2.

[0225] SF3B3.miniX on and SF3B3.X on have similar inductions in response to LMI070, but the protein expression level when using SF3B3.miniX on is significantly reduced compared to X on . Further studies were conducted to optimize the SF3B3.miniX cassette. Through rational design, the inventors optimized the Kozak / ATG translation initiation sequence and removed potential splicing sites within the intron sequence adjacent to the exon regulated by LMI070, generating novel miniXon sequences (SF3B3.miniXS3B and SF3B3.miniXS3B2). The following two modified cassette sequences were generated. on To optimize the SF3B3.miniX cassette, further studies were conducted. Through rational design, the inventors optimized the Kozak / ATG translation initiation sequence and removed potential splicing sites within the intron sequence adjacent to the exon regulated by LMI070, generating novel miniXon sequences (SF3B3.miniXS3B and SF3B3.miniXS3B2). The following two modified cassette sequences were generated.

[0226] SF3B3.miniXS3B (m3B; miniXS3B; XS3B): TIFF2025521120000006.tif48146

[0227] SF3B3.miniXS3B2 (m3B2; miniXS3B2; XS3B2): TIFF2025521120000007.tif48145

[0228] Provide further breakdowns of the arrays that make up SF3B3.miniXS3B and SF3B3.miniXS3B2 in Tables 3 and 4.

[0229] To test these switches, cassettes were cloned upstream of luciferase or cDNA. HEK293 cells were transfected with the candidate cassettes, treated with LMI070, and luciferase activity expression was measured after 24 hours. Increased luciferase expression was observed for each candidate cassette in response to LMI070, and the miniXS3B switch and the miniXS3B2 switch had improved induction compared to X on and miniX on and showed higher protein levels compared to the previous SF3B3.miniX on cassette (Figure 2).

[0230] Gene editing approaches offer a great opportunity to alter or remove disease alleles, but long-term expression of editing machinery from viral vectors can be problematic. Editing enzymes are foreign proteins and can induce an immune response, and long-term gene expression will increase the chance of off-target editing (Charlesworth et al., 2019; Vakulskas et al., 2018). To test the effectiveness of SF3B3.miXS3B and SF3B3.miXS3B2, which control the translation of CRISPR effector proteins, SF3B3.miXS3B-SaCas9-CRISPR expression cassettes and SF3B3.miXS3B2-SaCas9-CRISPR expression cassettes were generated, and SaCas9 protein levels in response to DMSO treatment or LMI070 treatment in HEK293 cells were quantified (Figure 3A). The SaCas9 protein was hardly detectable in cells treated with DMSO, but induction of the SaCas9 protein was evident after LMI070 treatment. In particular, the level of the SaCas9 protein was higher compared to the previous SF3B3.miniXon cassette when the translation of the SaCas9 protein was controlled by the miniXS3B cassette and the miniXS3B2 cassette.

[0231] miniX optimized to regulate editing onThe utility of the system was tested, as an example, using an allele-specific editing approach of mutant huntingtin (HTT) (Monteys et al., 2017), which is the target of a gene silencing approach for Huntington's disease (HD) (Tabrizi et al., 2019).

[0232] An allele-specific sgRNA sequence (PAM; sg935) using a 5'-single nucleotide polymorphism (SNP)-dependent PAM motif for mHTT exon 1 was used in combination with an sgRNA (sgi3) targeting the downstream intron. These gRNAs edit HTT exon 1 via SaCas9 and reduce the mRNA level and protein level of HTT (Monteys et al., 2017). For this purpose, a miniXS3B switch for drug-inducible SaCas9 expression was generated and compared with a constitutively active cassette and an SF3B3.Xon-SaCas9 cassette (Figure 4). Subsequently, SF3B3.miniXS3B-SaCas9 + related gRNAs were transfected into HEK293 cells, and the levels of the HTT locus, HTT mRNA, and HTT protein were evaluated. Upon LMI070 treatment, there was a simultaneous reduction at the RNA level, and the HTT transcript was reduced by 50% to the same extent as shown in cells transfected with a constitutively active editing expression cassette and an SF3B3.Xon-SaCas9 editing expression cassette (Figure 4A). The protein level was reduced similarly (Figure 4B). Collectively, these data indicate that the SF3B3.miniXS3B switch, together with allele-specific gRNAs for mHTT, represents an important advance in HD treatment.

[0233] (Table 1) SF3B3.X in the 5' to 3' direction on Sequence TIFF2025521120000008.tif161150*In exon 2, the Kozak sequence is shown in italics and the start codon is also underlined.

[0234] (Table 2) SF3B3.miniX in the 5' to 3' direction on Sequence TIFF2025521120000009.tif104150*In exon 2, the Kozak sequence is shown in italics and the start codon is also underlined.

[0235] (Table 3) SF3B3.miniXS3B sequence in the 5' to 3' direction TIFF2025521120000010.tif105150*In exon 2, the Kozak sequence is shown in italics and the start codon is also underlined.

[0236] (Table 4) SF3B3.miniXS3B2 sequence in the 5' to 3' direction TIFF2025521120000011.tif104150*In exon 2, the Kozak sequence is shown in italics and the start codon is also underlined.

[0237] All of the methods disclosed and claimed in this specification can be made and carried out without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the methods and steps of the methods or the order of the steps described herein without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain chemically or physiologically related agents can be used in place of the agents described herein and that the same or similar results will be achieved. All such similar alternatives and modifications that are obvious to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined in the appended claims.

[0238] References The following references are hereby specifically incorporated by reference herein to the extent that they provide details of exemplary procedures or other details that supplement the description herein. TIFF2025521120000012.tif184146TIFF2025521120000013.tif198146

Claims

[Claim 1] The invention described in the specification.

Images