Adeno-associated viral vectors for targeting deep brain structures

Abstract

Provided herein are targeting peptides and vectors comprising sequences encoding the targeting peptides that deliver agents to specific substructures in the brain. In particular, the targeting peptide is a component of a modified sequence-specific adeno-associated virus (AAV) capsid protein, and in addition, wherein the substructure of the brain may be a globular pallidum, a shell nucleus, an endocyst, a caudal nucleus, a screen nucleus, a nigra, a motor cortex, a brain island, a temporal lobe cortex, a thalamus, a hippocampus, a hypopus, and a deep cerebellar nucleus.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 340,823, filed on May 11, 2022, and U.S. Provisional Application No. 63 / 342,324, filed on May 16, 2022, the entire contents of each of which are hereby incorporated by reference.

[0003] References to sequence listings

[0004] This application contains a sequence listing XML that has been submitted electronically and is hereby incorporated by reference in its entirety. The sequence listing XML created on May 9, 2023 is named CHOPP0054WO_ST26.xml and is 32,221 bytes in size. Background Art 1. Technical Field

[0005] The present disclosure relates generally to the fields of medicine and virology. More specifically, the present disclosure relates to compositions and methods for delivering molecular therapeutics to deep brain structures.

[0006] 2. Description of Related Technology

[0007] Different strategies have been developed to produce AAV vector variants, including rational design and directed evolution. Rational design methods use the knowledge of AAV capsids to make targeted changes to the capsids to change transduction efficiency or specificity, such as tyrosine mutations on the capsid surface to improve transduction efficiency. However, there are no AAV variants that specifically or efficiently target deep brain structures. Diseases (including Huntington's Disease (HD) and Parkinson's Disease (PD)) mainly affect deep brain structures that existing AAV variants cannot effectively target. Therefore, AAV variants that can target deep brain structures are needed. Summary of the invention

[0008] Provided herein are viral vectors, each comprising a modified capsid, wherein the modified capsid comprises at least one amino acid sequence that targets the viral vector to a different brain structure.

[0009] In one embodiment, a modified adeno-associated virus (AAV) capsid protein is provided, which comprises a targeting peptide that targets a viral vector containing the modified AAV capsid protein to different organs or different brain structures, and the length of the targeting peptide is three to ten amino acids. In some aspects, the modified AAV capsid protein is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

[0010] In some aspects, the modified AAV capsid protein is derived from the AAV1 capsid protein (see SEQ ID NO: 1), and the targeting peptide is inserted after the 590th residue of the AAV1 capsid protein. In some aspects, the flank of the targeting peptide is a linker sequence, and the linker sequence on each side of the targeting peptide is two or three amino acids long. In some aspects, the linker sequence is SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide. In some aspects, the modified AAV1 capsid protein has a sequence with at least 95% identity to SEQ ID NO: 4.

[0011] In some aspects, the modified AAV capsid protein is derived from the AAV2 capsid protein (see SEQ ID NO: 2), and the targeting peptide is inserted after the 587th residue of the AAV2 capsid protein. In some aspects, the flank of the targeting peptide is a linker sequence, and the linker sequence on each side of the targeting peptide is two or three amino acids long. In some aspects, the linker sequence is AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide. In some aspects, the modified AAV2 capsid protein has a sequence with at least 95% identity to SEQ ID NO: 5.

[0012] In some aspects, the modified AAV capsid protein is derived from the AAV9 capsid protein (see SEQ ID NO:3), and the targeting peptide is inserted after the 588th residue of the AAV9 capsid protein. In some aspects, the flank of the targeting peptide is a linker sequence, and the linker sequence on each side of the targeting peptide is two or three amino acids long. In some aspects, the linker sequence is AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide. In some aspects, the modified AAV9 capsid protein has a sequence with at least 95% identity to SEQ ID NO:6.

[0013] In some aspects, the targeting peptide comprises a sequence of up to ten amino acids in length having an amino acid sequence selected from SEQ ID NOs: 7 to 13 or an amino acid sequence having at least 85% identity to an amino acid sequence selected from SEQ ID NOs: 8, 10, 12, 14, 16, and 18. In some aspects, the targeting peptide is seven amino acids in length. In some aspects, the AAV capsid protein has the sequence of any one of SEQ ID NOs: 19 to 24.

[0014] In some aspects, different brain structures are deep brain structures, such as globus pallidus, putamen, internal capsule, caudate nucleus, claustrum, substantia nigra, thalamus, hippocampus, subiculum and deep cerebellar nucleus. In some aspects, different brain structures are motor cortex or temporal lobe cortex. In some aspects, different brain structures are V / VI layer projection neurons in motor cortex and premotor cortex.

[0015] In one embodiment, provided herein is a nucleic acid comprising a sequence encoding the modified capsid protein of any one of the embodiments of the invention.

[0016] In one embodiment, provided herein is a recombinant adeno-associated virus (rAAV) comprising a modified capsid protein of any one of the embodiments of the invention. In some aspects, a combination of rAAVs is provided. In some aspects, rAAVs do not transduce hepatocytes.

[0017] In one embodiment, a viral vector is provided herein, comprising a nucleic acid encoding a modified capsid protein of any one of the embodiments of the present invention. In some aspects, the viral vector further comprises a nucleic acid sequence encoding a target nucleic acid. In some aspects, the target nucleic acid is a therapeutic agent. In some aspects, the therapeutic agent is an enzyme or an RNAi molecule.

[0018] In one embodiment, provided herein is a cell comprising a viral vector of any one of the embodiments of the invention. In some aspects, the cell is a mammalian cell, such as a human cell. In some aspects, the cell is in vitro or in vivo.

[0019] In one embodiment, provided herein is a pharmaceutical composition comprising a viral vector of an embodiment of the invention and a pharmaceutically acceptable carrier.

[0020] In one embodiment, provided herein are methods of delivering an agent to different brain structures of a subject comprising administering an AAV virus encoding the agent directly to the globus pallidus. In some aspects, the AAV virus is a virus of embodiments of the present invention.

[0021] In one embodiment, provided herein is a method for delivering an agent to different brain structures of an object, comprising administering a virus of an embodiment of the present invention to an object. In some aspects, different brain structures are globus pallidus, putamen, internal capsule, caudate nucleus, claustrum, substantia nigra, motor cortex, insula, temporal cortex, thalamus, hippocampus, subiculum, and deep cerebellar nucleus. In some aspects, different brain structures are projection neurons in layer V / VI of motor cortex and premotor cortex.

[0022] In some aspects, medicament is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein or CRISPR system. In some aspects, administration is administration to the central nervous system. In some aspects, administration is administration to the cerebellomedullary cistern, intraventricular space, ependyma, ventricle, subarachnoid space and / or intrathecal space. In some aspects, ventricle is anterior lateral ventricle (rostral lateral ventricle), and / or posterior lateral ventricle (caudal lateralventricle), and / or right lateral ventricle, and / or left lateral ventricle, and / or right anterior lateral ventricle, and / or left anterior lateral ventricle, and / or right posterior lateral ventricle, and / or left posterior lateral ventricle. In some aspects, administration is systemic.

[0023] In some aspects, a plurality of viral particles are administered. In some aspects, the virus is administered at a concentration of about 1×10 6 About 1×10 18 In some aspects, the virus is administered at a dose of about 1×10 7 Up to 1×10 17 , about 1×10 8 Up to 1×10 16 , about 1×10 9 Up to 1×10 15 , about 1×10 10 Up to 1×10 14 , about 1×10 10 Up to 1×10 13 , about 1×10 10 Up to 1×10 13 , about 1×10 10 Up to 1×10 11 , about 1×10 11 Up to 1×10 12 , about 1×10 12 Up to ×10 13 , or about 1×10 13 Up to 1×10 14 vg / kg patient. In some aspects, the subject is a human.

[0024] In one embodiment, provided herein is a method for treating a disease in a mammal, comprising administering a virus of an embodiment of the present invention to a mammal. In some aspects, the disease is a neurodegenerative disease. In some aspects, the neurodegenerative disease is Huntington's disease, amyotrophic lateral sclerosis (ALS), hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, Alzheimer's disease, polyglutamine repeat disease, or Parkinson's disease. In some aspects, the mammal is a human.

[0025] According to the following detailed description, other objects, features and advantages of the present invention will become apparent. However, it should be understood that although the detailed description and specific examples show some preferred embodiments of the present invention, they are only given by way of illustration, because according to the detailed description, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings constitute part of this specification and are included to further illustrate certain aspects of the present invention. The present invention may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments shown herein.

[0027] Figure 1 . Fluorescent In Situ Hybridization (FISH) images of each specific fluorophore used to localize mRNA within cells of the globus pallidus (injection site) (captured by confocal microscopy). Positive signals were localized to the cell bodies and nuclei of these cells. Most cells were positive for all three capsid variants.

[0028] Figure 2 . Epifluorescence images for locating proteins from each specific fluorophore in globus pallidus (injection site) cells. Positive signals are localized to both the cell bodies and processes of these cells. Specifically, although mRuby3 mRNA is present in the cell bodies of globus pallidus neurons by FISH, there is almost no positive signal for protein in the slab of tissue.

[0029] Figure 3 .From and Figure 2 The images in the epifluorescence images of different globus pallidus regions localize the proteins from each specific fluorophore within the cells of the posterior globus pallidus (injection site). The positive signal is localized to both the cell bodies and processes of these cells. In this tissue section, the mRuby signal is present.

[0030] Figure 4 Fluorescence in situ hybridization (FISH) images of each specific fluorophore used to localize mRNA within putamen cells (captured by confocal microscopy). Positive mRNA signals were localized to the cell bodies and nuclei of these cells. The capsid variant expressing mNeonGreen (mNG) showed the highest cell transduction in the putamen.

[0031] Figure 5 .Epifluorescence images used to localize proteins from each specific fluorophore within putamen cells. Positive signals were localized to both the soma and processes of these cells. Similar to mRNA by FISH, the new capsid variant expressing mNeonGreen (mNG) showed the most positive signals in both cells and processes.

[0032] Figure 6 Epifluorescence images used to localize proteins from each specific fluorophore within the internal vesicle. The positive signals from mTFP1 and NeonGreen fluorescence are clearly separated.

[0033] Figure 7 Fluorescence in situ hybridization (FISH) images of each specific fluorophore used to localize mRNA within caudate nucleus cells, which were captured by confocal microscopy. Positive mRNA signals were localized to the cell bodies of these cells.

[0034] Figure 8 Epifluorescence images used to localize proteins from each specific fluorophore within caudate nucleus cells. Positive signals were localized to both the cell bodies and processes of these cells.

[0035] Fig. 9 Fluorescence in situ hybridization (FISH) images of each specific fluorophore used to localize mRNA within claustrum cells, which were captured by confocal microscopy. Positive mRNA signals were localized to the cell bodies of these cells.

[0036] Fig.10 .Epifluorescence images used to localize proteins from each specific fluorophore within the claustrum. Again, positive fluorescence signals were observed in both cell bodies and cell processes.

[0037] Fig.11 Fluorescence in situ hybridization (FISH) images of each specific fluorophore used to localize mRNA within substantia nigra cells, which were captured by confocal microscopy. Positive mRNA signals were localized to the cell bodies and nuclei of these cells.

[0038] Fig.12Fluorescence in situ hybridization (FISH) images of each specific fluorophore used to localize mRNA within substantia nigra cells (captured by confocal microscopy), but relative to Fig.11 The images in the figure are from different regions. Positive mRNA signals are localized to the cell bodies and nuclei of these cells.

[0039] Fig.13 .Epifluorescence images used to localize proteins from each specific fluorophore within the substantia nigra. Positive fluorescent signals were observed in both cell bodies and cell processes.

[0040] Fig.14 .Epifluorescence images used to localize proteins from each specific fluorophore within the substantia nigra, but relative to Fig.13 The images in FIG. 1 are from different regions. Positive fluorescence signals were observed in both cell bodies and cell processes.

[0041] Fig.15 Fluorescence in situ hybridization (FISH) images of each specific fluorophore used to localize mRNA within motor cortex cells, which were captured by confocal microscopy. Positive mRNA signals were localized to the cell bodies and nuclei of these cells.

[0042] Fig.16 Epifluorescence images used to localize proteins from each specific fluorophore within motor cortical cells. Positive signals were localized to both the soma and processes of these cells. Strikingly, both mTFP1 and mNeonGreen presented mRNA signals by FISH; however, mTFP1 was predominantly expressed as shown by epifluorescence.

[0043] Fig.17 In the cingular cortex, a few positive cells were detected by FISH for the new capsid mRNA, whereas widespread mTFP1-positive cells were detected by epifluorescence microscopy.

[0044] Fig.18 Fluorescence in situ hybridization (FISH) images of each specific fluorophore used to localize mRNA within insular cells of the temporal cortex (captured by confocal microscopy). Positive mRNA signals were localized to the cell bodies (cytoplasm and nucleus) of these cells.

[0045] Fig.19 Epifluorescence images used to localize proteins from each specific fluorophore within insular cells of the temporal cortex. Positive signals were localized to both the somata and processes of these cells.

[0046] Fig. 20. Fluorescence in situ hybridization (FISH) images of each specific fluorophore used to localize mRNA within thalamic cells (captured by confocal microscopy). Positive mRNA signals were localized to the cell bodies (cytoplasm and nucleus) of these cells.

[0047] Fig.21 .Epifluorescence images used to localize proteins from each specific fluorophore within thalamic cells. Positive signals were localized to both the somata and processes of these cells.

[0048] Figure 22A to D. Epifluorescence images of the first three new capsids delivered to the globus pallidus of rhesus macaques. Expression of all three was high in the putamen. A) Image showing expression of AAV1.THTD.mTFP1; B) Image showing expression of AAV2.SGGR.mNeonGreen; C) Image showing expression of AAV2.SKGQ.mRuby3; D) Merged image of A to C.

[0049] Figure 23A to D. Epifluorescence images of the first three new capsids delivered to the globus pallidus of rhesus monkeys. Expression of AAV1.THTD.mTFP1 is extremely high in the motor cortex. A) Image showing expression of AAV1.THTD.mTFP1; B) Image showing expression of AAV2.SGGR.mNeonGreen; C) Image showing expression of AAV2.SKGQ.mRuby3; D) Merged image of A to C.

[0050] Figure 24A to D. Epifluorescence images of the first three new capsids delivered to the globus pallidus of rhesus monkeys. Expression of all three is localized in the hippocampus (CA1) and adjacent subiculum. A) Image showing expression of AAV1.THTD.mTFP1; B) Image showing expression of AAV2.SGGR.mNeonGreen; C) Image showing expression of AAV2.SKGQ.mRuby3; D) Merged image of A to C.

[0051] Fig.25 Fluorescence in situ hybridization (FISH) images of each specific fluorophore used to localize mRNA within cells of the globus pallidus (injection site) of a second rhesus monkey (captured by confocal microscopy). Positive signals were localized to the cell bodies and nuclei of these cells.

[0052] Fig.26 Epifluorescence images used to localize proteins from each specific fluorophore within cells of the globus pallidus (injection site) of a second rhesus monkey. Positive signals were localized to both the somata and processes of these cells.

[0053] Fig. 27Wholemounts of brain and spinal cord from mice injected with virus into the internal capsule / globus pallidus. Mice are numbered on the left, and their ID numbers are color-coded to reflect which hemisphere received which fluorophore-expressing capsid.

[0054] Fig.28 Whole mounts of brain and spinal cord from mice injected with virus into the thalamus. Mice are numbered on the left, and their ID numbers are color-coded to reflect which hemisphere received which capsid expressing the fluorophore.

[0055] Fig.29 Whole mounts of brain and spinal cord from mice injected with virus into deep cerebellar nuclei. Mice are numbered on the left, and their ID numbers are color-coded to reflect which hemisphere received which fluorophore-expressing capsid.

[0056] Fig.30 Four coronal brain sections from mice injected with AAV2.SGGR.mNeonGreen into the left internal capsule and medial globus pallidus and AAV2.SKGO.mRuby3 into the right side.

[0057] Fig.31 .Three coronal brain sections from mice injected with AAV2.SKGO.mRuby3 into the left internal capsule and medial globus pallidus and AAV1.THTD.mTFP1 into the right side.

[0058] Fig.32 Four coronal brain sections from a mouse injected with AAV2.SGGR.mNeonGreen into the left thalamus and AAV2.SKGO.mRuby3 into the right. Single sagittal section of the cerebellum and hindbrain.

[0059] Fig.33 Four coronal brain sections from a mouse injected with AAV2.SKGO.mRuby3 into the left thalamus and AAV1.THTD.mTFP1 into the right side. Sagittal sections of the cerebellum and brainstem show that neurons in the deep cerebellar nuclei are positive for AAV1.THTD.mTFP1.

[0060] Fig.34 Sagittal sections of the cerebellum in which AAV1.THTD.mTFP1 was injected into the left deep cerebellar nucleus and AAV2.SGGR.mNeonGreen was injected into the right.

[0061] Fig.35 Sagittal sections of the cerebrum in which AAV1.THTD.mTFP1 was injected into the left deep cerebellar nucleus and AAV2.SGGR.mNeonGreen was injected into the right lateral cerebrum.

[0062] Fig.36 Sagittal sections of the whole brain with AAV1.THTD.mTFP1 injected into the left deep cerebellar nucleus and AAV2.SGGR.mNeonGreen injected into the right. Neurons and processes throughout the brain were positive for AAV1.THTD.mTFP1.

[0063] Fig.37 Sagittal sections of the whole brain with AAV2.SKGO.mRuby3 injected into the left deep cerebellar nucleus and AAV1.THTD.mTFP1 injected into the right. Neurons and processes throughout the brain were positive for AAV1.THTD.mTFP1.

[0064] Figures 38A to 38C Heatmap representation of UMI enrichment of AAV capsid variants in sampled brain regions. Fig.38A )Amplicon seq spanning the region where the AAV peptide was inserted allowed us to collect unique molecular identifier (UMI) counts for capsid variants delivered in many sampled tissues. Amplicons in A were generated from capsid DNA recovered from transduced tissues. The % abundance of each capsid variant is representative of each specified tissue. Capsids that were fluorescently validated are shown. ( Fig.38B ) The same DNA Amplicon-Seq UMI counts shown in A were normalized by calculating enrichment relative to the input. The most highly represented capsid candidates are those that show the highest enrichment relative to their abundance in the input capsid pool. ( Fig.38C ) % abundance of UMI counts obtained from RNA by Amplicon-Seq.

[0065] Fig.39 .Epifluorescence images of AAV1.THTD.mTFP1 localized to a large number of layer V / VI cortical projection neurons in the motor and premotor cortices of rhesus monkeys.

[0066] Fig.40 .AAV gDNA and protein expression was below the detection threshold in the liver of rhesus macaques. DETAILED DESCRIPTION

[0067] Provided herein are viral vectors, each of which comprises a modified capsid, wherein the modified capsid comprises at least one amino acid sequence that targets the viral vector to a deep brain structure. In certain embodiments, the brain structure is the globus pallidus, putamen, internal capsule, caudate nucleus, claustrum, substantia nigra, motor cortex, insula, temporal cortex, thalamus, hippocampus, subiculum, and deep cerebellar nucleus. In certain embodiments, AAV is AAV1, AAV2, or AAV9. An exemplary wild-type reference AAV1 capsid protein sequence is provided in SEQ ID NO: 1. An exemplary wild-type reference AAV2 capsid protein sequence is provided in SEQ ID NO: 2. An exemplary wild-type reference AAV9 capsid protein sequence is provided in SEQ ID NO: 3. In certain aspects, the targeting peptide is inserted at position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, or position 588 of the AAV9 capsid. An exemplary modified AAV1 capsid protein sequence is provided in SEQ ID NO:4, which shows that the targeting peptide insertion after position 590 is SSAX7AS, wherein the front SSA and the tail AS are linker sequences and X7 represents the targeting peptide. An exemplary modified AAV2 capsid protein sequence is provided in SEQ ID NO:5, which shows that the targeting peptide insertion after position 587 is AAAX7AA, wherein the front AAA and the tail AA are linker sequences and X7 represents the targeting peptide. An exemplary modified AAV9 capsid protein sequence is provided in SEQ ID NO:6, which shows that the targeting peptide insertion after position 588 is AAAX7AS, wherein the front AAA and the tail AS are linker sequences and X7 represents the targeting peptide.

[0068] I. Adeno-associated virus (AAV) vector

[0069] Adeno-associated virus (AAV) is a small, non-pathogenic virus of the Parvoviridae family. To date, many serologically different AAVs have been identified, and more than a dozen have been isolated from humans or primates. AAV differs from other members of the family in that it relies on a helper virus for replication.

[0070] The AAV genome can exist in an extrachromosomal state without integrating into the genome of the host cell; has a wide host range; transduces both dividing and non-dividing cells in vitro and in vivo, and maintains high levels of expression of transduced genes. AAV virus particles are thermostable; are resistant to solvents, detergents, pH changes and temperature; and can be column purified and / or concentrated on a CsCl gradient or by other means. The AAV genome contains positive or negative single-stranded deoxyribonucleic acid (ssDNA). The approximately 4.7kb genome of AAV consists of a segment of single-stranded DNA at the positive or negative pole. At the end of the genome are short reverse terminal repeats (ITRs), which can be folded into a hairpin structure and used as the starting point for viral DNA replication.

[0071] AAV "genome" refers to the recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form AAV particles. AAV particles typically contain an AAV genome packaged with AAV capsid proteins. In the case of using a recombinant plasmid to construct or prepare a recombinant vector, the AAV vector genome does not contain a portion of a "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called a "plasmid backbone," which is important for plasmid cloning and amplification (a process required for plasmid proliferation and production), but is not itself packaged or encapsulated in viral particles. Therefore, the AAV vector "genome" refers to a nucleic acid packaged or encapsulated by an AAV capsid protein.

[0072] AAV virion (particle) is a non-enveloped icosahedral particle with a diameter of about 25nm containing an AAV capsid. AAV particles contain icosahedral symmetry, which is composed of three related capsid proteins (VP1, VP2 and VP3), which interact to form capsids. The genome of most natural AAVs usually contains two open reading frames (ORF), sometimes referred to as left ORF and right ORF. Right ORF usually encodes capsid proteins VP1, VP2 and VP3. These proteins are usually present in a ratio of 1:1:10, but can exist in different ratios and all derive from right ORF. VP1, VP2 and VP3 capsid proteins are different from each other by using alternative splicing and unique start codons. Deletion analysis shows that the removal or change of VP1 translated from the information of alternative splicing leads to a reduced yield of infectious particles. Mutations in the VP3 coding region result in the inability to produce any single-stranded progeny DNA or infectious particles. In certain embodiments, the genome of AAV particles encodes one, two or all three VP1, VP2 and VP3 polypeptides.

[0073] The left ORF usually encodes nonstructural Rep proteins, Rep 40, Rep 52, Rep 68 and Rep 78, 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 related to the preferential integration of the AAV genome into the region of the q arm of human chromosome 19. It has been shown that Rep68 / 78 has NTP binding activity and DNA and RNA helicase activity. Some Rep proteins have nuclear localization signals and 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 are therefore not included in the AAV particles containing nucleic acids encoding polypeptides.

[0074] The end of the AAV genome contains short reverse terminal repeats (ITRs), which have the potential to fold into a T-type hairpin structure, which is used as the starting point for viral DNA replication. Therefore, the genome of AAV contains one or more ITR sequences (e.g., ITR sequence pairs), which are located on the flank of the single-stranded viral DNA genome. The length of the ITR sequence is generally about 145 bases each. In the ITR region, two elements that are considered to be the core of ITR function, the GAGC repeat motif and the terminal resolution site (trs), have been described. When ITR is in a linear or hairpin conformation, the repeat motif shows binding to Rep. The combination is considered to be locating Rep68 / 78 at trs for cutting, which occurs in a site-specific and strand-specific manner. In addition to their role in replication, these two elements also show that they are the core of viral integration. Contained in the chromosome 19 integration locus is a Rep binding site with adjacent trs. It has been shown that these elements are functional and are essential for locus-specific integration.

[0075] The term "recombinant" as a modifier of vectors (e.g., recombinant viruses, such as lentiviral or parvoviral (e.g., AAV) vectors) and sequence modifiers (e.g., recombinant nucleic acid sequences and polypeptides) means that the composition has been manipulated (i.e., modified) in a manner that does not normally occur in nature. A specific example of a recombinant vector (e.g., AAV, retroviral, or lentiviral vector) is the insertion of a nucleic acid sequence that is not normally present in the wild-type viral genome into the viral genome. An example of a recombinant nucleic acid sequence is a nucleic acid (e.g., a gene) encoding an inhibitory RNA cloned into a vector with or without the 5', 3', and / or intronic regions that are normally associated with the gene in the viral genome. Although the term "recombinant" is not always used herein to refer to vectors (e.g., viral vectors) and sequences (e.g., polynucleotides), "recombinant" forms including nucleic acid sequences, polynucleotides, transgenes, etc. are explicitly included despite any such omissions.

[0076] Recombinant virus "vector" is derived from the wild-type genome of the virus, by using molecular methods to remove a part of the wild-type genome from the virus, and replace it with a non-natural nucleic acid (e.g., a nucleic acid sequence). Generally speaking, for example, for AAV, one or two reverse terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. "Recombinant" viral vectors (e.g., rAAV) are different from viral (e.g., AAV) genomes because a part of the viral genome has been replaced by non-natural sequences for viral genomic nucleic acids (e.g., nucleic acids encoding transactivators or nucleic acids encoding inhibitory RNAs or nucleic acids encoding therapeutic proteins). Therefore, the incorporation of such non-natural nucleic acid sequences defines viral vectors as "recombinant" vectors, which can be referred to as "rAAV vectors" in the case of AAV.

[0077] In certain embodiments, AAV (e.g., rAAV) comprises two ITRs. In certain embodiments, AAV (e.g., rAAV) comprises an ITR pair. In certain embodiments, AAV (e.g., rAAV) comprises an ITR pair flanking (i.e., at each 5' and 3' end) at least a nucleic acid sequence encoding a polypeptide having a function or activity.

[0078] AAV vectors (e.g., rAAV vectors) can be packaged and referred to herein as "AAV particles" for subsequent infection (transduction) of cells in vitro, in vitro, or in vivo. In the case where a recombinant AAV vector is encapsulated or packaged into an AAV particle, the particle may also be referred to as a "rAAV particle". In certain embodiments, the AAV particle is a rAAV particle. The rAAV particle typically comprises an rAAV vector or a portion thereof. The rAAV particle may be one or more rAAV particles (e.g., a plurality of AAV particles). The rAAV particle typically comprises a protein (e.g., a capsid protein) that encapsulates or packages the rAAV vector genome. It should be noted that reference to rAAV vectors may also be used to refer to rAAV particles.

[0079] Any suitable AAV particles (e.g., rAAV particles) can be used for the methods or purposes herein. rAAV particles and / or the genomes contained therein can be derived from any suitable serotype or strain of AAV. rAAV particles and / or the genomes contained therein can be derived from two or more serotypes or strains of AAV. Therefore, rAAV can comprise proteins and / or nucleic acids or parts thereof of any serotype or strain of AAV, wherein the AAV particles are suitable for infection and / or transduction of mammalian cells. Some 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.

[0080] In certain embodiments, a plurality of rAAV particles comprise particles of the same strain or serotype (or subgroup or variant), or are derived from the same strain or serotype (or subgroup or variant). In certain embodiments, a plurality of rAAV particles comprise a mixture of two or more different rAAV particles (e.g., different serotypes and / or strains).

[0081] As used herein, the term "serotype" is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological uniqueness is determined based on the lack of cross-reactivity between antibodies to one AAV and antibodies to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2 and / or VP3 sequence differences between AAV serotypes). Although there is the possibility that AAV variants (including capsid variants) may not be serologically distinguishable from reference AAV or other AAV serotypes, they differ in at least one nucleotide or amino acid residue compared to the reference AAV or other AAV serotypes.

[0082] In certain embodiments, the rAAV vector based on the first serotype genome corresponds to the serotype of one or more capsid proteins that package the vector. For example, the serotype of one or more AAV nucleic acids (e.g., ITRs) comprising the AAV vector genome corresponds to the serotype of the capsid that comprises the rAAV particle.

[0083] In certain embodiments, the rAAV vector genome may be based on an AAV (e.g., AAV2) serotype genome that is different from the serotype of one or more AAV capsid proteins that package the vector. For example, the rAAV vector genome may comprise an AAV2-derived nucleic acid (e.g., ITR), while at least one or more of the three capsid proteins are derived from a different serotype, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype or variants thereof.

[0084] In certain embodiments, the rAAV particles or their vector genomes associated with a reference serotype have a polynucleotide, polypeptide, or subsequence thereof comprising or consisting of a sequence that is at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a polynucleotide, polypeptide, or subsequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 particle. In some specific embodiments, the rAAV particles or their vector genomes associated with a reference serotype have a capsid or ITR sequence comprising or consisting of a sequence that is at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a capsid or ITR sequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype.

[0085] In certain embodiments, the methods herein comprise the use, administration, or delivery of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74, or rAAV-2i8 particles.

[0086] In certain embodiments, the method herein includes using, applying or delivering rAAV2 particles. In certain embodiments, rAAV2 particles include AAV2 capsids. In certain embodiments, rAAV2 particles include one or more capsid proteins (e.g., VP1, VP2 and / or VP3), and the one or more capsid proteins have at least 60%, 65%, 70%, 75% or higher homogeneity with the corresponding capsid proteins of natural or wild-type AAV2 particles, for example, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% homogeneity. In certain embodiments, the rAAV2 particle comprises VP1, VP2, and VP3 capsid proteins that are at least 75% identical or more to the corresponding capsid proteins of a native or wild-type AAV2 particle, e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical. In certain embodiments, the rAAV2 particle is a variant of a native or wild-type AAV2 particle. In some aspects, one or more capsid proteins of the AAV2 variant have 1, 2, 3, 4, 5, 5 to 10, 10 to 15, 15 to 20 or more amino acid substitutions compared to the capsid protein of a native or wild-type AAV2 particle.

[0087] In certain embodiments, the rAAV9 particles include an AAV9 capsid. In certain embodiments, the rAAV9 particles include one or more capsid proteins (e.g., VP1, VP2, and / or VP3) that are at least 60%, 65%, 70%, 75% or more identical to the corresponding capsid proteins of native or wild-type AAV9 particles, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical. In certain embodiments, the rAAV9 particle comprises VP1, VP2, and VP3 capsid proteins that are at least 75% identical to the corresponding capsid proteins of a native or wild-type AAV9 particle, e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical. In certain embodiments, the rAAV9 particle is a variant of a native or wild-type AAV9 particle. In some aspects, one or more capsid proteins of the AAV9 variant have 1, 2, 3, 4, 5, 5 to 10, 10 to 15, 15 to 20 or more amino acid substitutions compared to the capsid protein of a native or wild-type AAV9 particle.

[0088] In certain embodiments, the rAAV particle comprises one or two ITRs (e.g., an ITR pair) that have at least 75% identical sequence to the corresponding ITR of native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, or AAV-2i8. %, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identity, so long as it retains one or more desired ITR functions (e.g., the ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into the host cell genome; and / or packaging, if desired).

[0089] In certain embodiments, the rAAV2 particle comprises one or two ITRs (e.g., an ITR pair) that are at least 75% or more identical to the corresponding ITRs of a native or wild-type AAV2 particle, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical, so long as it retains one or more desired ITR functions (e.g., the ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into the host cell genome; and / or packaging, if desired).

[0090] In certain embodiments, the rAAV9 particle comprises one or two ITRs (e.g., an ITR pair) that are at least 75% or more identical to the corresponding ITRs of a native or wild-type AAV2 particle, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identity, so long as it retains one or more desired ITR functions (e.g., the ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into the host cell genome; and / or packaging, if desired).

[0091] rAAV particles may comprise ITRs having any suitable number of "GAGC" repeats. In certain embodiments, the ITRs of AAV2 particles comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more "GAGC" repeats. In certain embodiments, rAAV2 particles comprise ITRs containing three "GAGC" repeats. In certain embodiments, rAAV2 particles comprise ITRs having less than four "GAGC" repeats. In certain embodiments, rAAV2 particles comprise ITRs having more than four "GAGC" repeats. In certain embodiments, the ITRs of rAAV2 particles comprise a Rep binding site in which the fourth nucleotide in the first two "GAGC" repeats is C instead of T.

[0092] An exemplary suitable length of DNA that can be incorporated into a rAAV vector for packaging / encapsulation into rAAV particles can be about 5 kilobases (kb) or less. In some specific 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.

[0093] The rAAV vector comprising a nucleic acid sequence for guiding the expression of RNAi or polypeptides can be produced using suitable recombinant techniques known in the art (e.g., see Sambrook et al., 1989). Recombinant AAV vectors are usually packaged into AAV particles with transduction ability and propagated using the AAV virus packaging system. AAV particles with transduction ability can bind to and enter mammalian cells, and then deliver nucleic acid carriers (e.g., heterologous genes) to the nucleus of cells. Therefore, complete rAAV particles with transduction ability are configured to transduce mammalian cells. The rAAV particles configured to transduce mammalian cells generally do not have replication ability, and require additional protein mechanisms to replicate themselves. Therefore, the rAAV particles configured to transduce mammalian cells are modified to bind to and enter mammalian cells, and nucleic acid is delivered to cells, wherein the nucleic acid for delivery is generally located between the AAV ITR pairs in the rAAV genome.

[0094] Suitable host cells for producing transduction-capable AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can or have been used as recipients of heterologous rAAV vectors. Cells from a stable human cell line HEK293 (easily obtained by, for example, the American Type Culture Collection with accession number ATCC CRL1573) can be used. In certain embodiments, a modified human embryonic kidney cell line (e.g., HEK293) transformed with type 5 adenovirus DNA fragments and expressing adenovirus E1a and E1b genes is used to produce recombinant AAV particles. The modified HEK293 cell line is easily transfected and provides a particularly convenient platform for producing rAAV particles therein. Methods for producing high-titer AAV particles capable of transducing mammalian cells are known in the art. For example, AAV particles can be prepared as shown in Wright, 2008 and Wright, 2009.

[0095] In certain embodiments, before or simultaneously with transfection of the AAV expression vector, the AAV auxiliary function is introduced into the host cell by transfecting the host cell with an AAV auxiliary construct. Therefore, the AAV auxiliary construct is sometimes used to provide at least transient expression of the AAV rep and / or cap genes to supplement the AAV function of the deletion necessary for productive AAV transduction. The AAV auxiliary construct generally lacks the AAV ITR and can neither replicate nor package itself. These constructs can be in the form of plasmids, phages, transposons, clays, viruses or virions. Many AAV auxiliary constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 encoding both Rep and Cap expression products. Many other vectors encoding Rep and / or Cap expression products are known.

[0096] An "expression vector" is a specialized vector containing a gene or nucleic acid sequence with the necessary regulatory regions required for expression in a host cell. An expression vector may contain at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous nucleic acid sequence, expression control elements (e.g., promoters, enhancers), introns, ITRs, and polyadenylation signals.

[0097] II. Therapeutic Agents

[0098] In some embodiments, viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding inhibitory RNA, noncoding RNA and / or therapeutic proteins to cells in culture or in a host organism.

[0099] A. Inhibitory RNA

[0100] "RNA interference (RNAi)" is a process of sequence-specific post-transcriptional gene silencing initiated by siRNA. During RNAi, siRNA induces degradation of target mRNA and subsequently leads to sequence-specific inhibition of gene expression.

[0101] "Inhibitory RNA", "RNAi", "small interfering RNA" or "short interfering RNA" or "siRNA" molecules, "short hairpin RNA" or "shRNA" molecules, or "miRNA" are RNA duplexes that target nucleotides of a target nucleic acid sequence. The term "siRNA" as used herein is a general term that covers a subset of shRNA and miRNA. "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule. siRNA "targets" a gene because the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the target gene. In certain embodiments, the siRNA targets a sequence encoding huntingtin. In some embodiments, the length of the duplex of the siRNA is less than 30 base pairs. In some embodiments, the length of 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 some embodiments, the length of the duplex is 19 to 25 base pairs in length. In certain embodiments, the length of the duplex is 19 or 21 base pairs in length. The RNA duplex portion of the siRNA can be a part of a hairpin structure. In addition to the duplex portion, the hairpin structure can include a loop portion between the two sequences that form the duplex. The length of the loop can vary. In some embodiments, the length of 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 certain embodiments, the length of the loop is 18 nucleotides. The hairpin structure can also include 3' and / or 5' overhangs. In some embodiments, the overhang is a 3' and / or 5' overhang of 0, 1, 2, 3, 4 or 5 nucleotides in length.

[0102] shRNA is composed of a stem-loop structure, which is designed to include a 5' flanking region, a siRNA region segment, a loop region, a 3' siRNA region, and a 3' flanking region. Most RNAi expression strategies utilize short-hairpin RNA (shRNA) driven by a strong polIII-based promoter. Many shRNAs show effective knockdown of target sequences in vitro and in vivo, however, some shRNAs that show effective knockdown of target genes are also found to be toxic in vivo.

[0103] miRNA is a small cellular RNA (about 22nt) processed from a precursor stem-loop transcript. Known miRNA stem-loops can be modified to contain RNAi sequences that are specific to the target gene. MiRNA molecules can be superior to shRNA molecules because miRNA is endogenously expressed. Therefore, miRNA molecules are less likely to induce the interferon pathway of dsRNA response, they are more effectively processed than shRNA, and have been shown to be 80% more effective in silencing.

[0104] A recently discovered alternative is to use artificial miRNA (pri-miRNA scaffolds that shuttle siRNA sequences) as RNAi vectors. Artificial miRNAs are more naturally similar to endogenous RNAi substrates and are more susceptible to Pol-II transcription (e.g., allowing tissue-specific expression of RNAi) and polycistronic strategies (e.g., allowing delivery of multiple siRNA sequences). See U.S. Patent No. 10,093,927, which is incorporated by reference.

[0105] The transcription unit of "shRNA" consists of a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides. shRNA is exported from the nucleus by export protein-5, and once in the cytoplasm, it is processed by Dicer to produce functional siRNA. "miRNA" stem-loop consists of a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides, which is usually expressed as part of a larger primary transcript (pri-miRNA), which is excised by the Drosha-DGCR8 complex to produce an intermediate product called precursor miRNA, which is then exported from the nucleus by export protein-5, and once in the cytoplasm, it is processed by Dicer to produce functional siRNA. "Artificial miRNA" or "artificial miRNA shuttle vector" used herein refers interchangeably to a primary miRNA transcript having a region of a duplex stem loop (at least about 9 to 20 nucleotides), which is excised by Drosha and Dicer processing and replaced by the siRNA sequence of the target gene, while retaining the structural elements within the stem loop necessary for effective Drosha processing. The term "artificial" originates 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. The term "miRNA" as used herein encompasses both naturally occurring miRNA sequences as well as artificially generated miRNA shuttle vectors.

[0106] The siRNA may be encoded by a nucleic acid sequence, and the nucleic acid sequence may further comprise a promoter. The nucleic acid sequence may further comprise a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a sequence of six Ts.

[0107] In designing RNAi, several factors need to be considered, such as the properties of siRNA, the persistence of silencing and the selection of delivery systems. In order to produce the RNAi effect, the siRNA introduced into the organism will usually contain exon sequences. In addition, the RNAi process is homology-dependent, so the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous sequences rather than gene-specific sequences. Preferably, siRNA shows greater than 80%, 85%, 90%, 95%, 98% or even 100% identity between the sequence of siRNA and the gene sequence to be inhibited. Sequences with less than about 80% identity to the target gene are not so effective in fact. Therefore, the greater the homology between siRNA and the gene to be inhibited, the less likely the expression of unrelated genes will be affected.

[0108] In addition, the size of siRNA is an important consideration. In some embodiments, the present invention relates to siRNA molecules comprising at least about 19 to 25 nucleotides and capable of regulating gene expression. In the context of the present invention, the length of siRNA is preferably less than 500, 200, 100, 50 or 25 nucleotides. More preferably, the length of siRNA is about 19 nucleotides to about 25 nucleotides.

[0109] siRNA target generally means a polynucleotide comprising a region encoding a polypeptide or a polynucleotide region regulating replication, transcription, or translation or other processes important to polypeptide expression; or a polynucleotide comprising a region encoding a polypeptide and a region operably linked thereto regulating expression. Any gene expressed in a cell can be targeted. Preferably, the target gene is a gene related to a cell activity progression that is important for a disease or is particularly interesting as a research subject or is associated with the cell activity progression.

[0110] B. Non-coding RNA

[0111] As demonstrated by cDNA cloning projects and genomic tile arrays, more than 90% of the human genome undergoes transcription but does not encode proteins. These transcription products are called non-protein coding RNA (ncRNA). Various ncRNA transcripts, such as ribosomal RNA, transfer RNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA), are essential for cell function. Similarly, a large number of short ncRNAs, such as microRNA (miRNA), endogenous short interfering RNA (siRNA), PIWI-interacting RNA (piRNA) and small nucleolar RNA (snoRNA), are also known to play important regulatory roles in eukaryotic cells. Recent studies have shown that the group of long ncRNA (lncRNA) transcripts exhibits cell type-specific expression and is located in specific subcellular compartments. lncRNAs are also known to play important roles during cell development and differentiation, supporting the idea that they were selected during the process of evolution.

[0112] lncRNAs have been shown to have many different functions. In many cases, they have been shown to play a role in regulating the activity or localization of proteins, or to serve as an organizing framework for subcellular structures. In other cases, lncRNAs are processed to produce a variety of small RNAs, or they may regulate how other RNAs are processed. The latest version of data (version #27) produced by the public research consortium GenCode catalogued just under 16,000 lncRNAs in the human genome, yielding nearly 28,000 transcripts; when other databases are included, more than 40,000 lncRNAs are known.

[0113] Interestingly, lncRNAs can affect the expression of specific target proteins at specific genomic loci, modulate the activity of protein-binding partners, direct chromatin-modifying complexes to their sites of action, and be post-transcriptionally processed to generate a large number of 5'-capped small RNAs. Epigenetic pathways can also regulate the differential expression of lncRNAs.

[0114] There is also increasing evidence that abnormally expressed lncRNAs play an important role in normal physiological processes and in a variety of disease states. lncRNAs are dysregulated in a variety of diseases, including ischemia, heart disease, Alzheimer's disease, psoriasis, and spinocerebellar ataxia type 8. This dysregulation is also manifested in a variety of cancers, such as breast cancer, colon cancer, prostate cancer, hepatocellular carcinoma, and leukemia. Several lncRNAs (e.g., gadd74 and lncRNA-RoR5) regulate cell cycle regulators such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and p53, and thus provide additional flexibility and robustness layers for cell cycle progression. In addition, some lncRNAs are associated with mitotic processes, such as centromeric satellite RNAs, which are essential for kinetochore formation and are therefore essential for chromosome segregation during mitosis in humans and Drosophila. Another nuclear lncRNA, MA-lincl, regulates M phase exit by functioning in cis to repress the expression of its neighboring gene Pura, a regulator of cell proliferation.

[0115] lncRNA is a group of transcripts generally defined as more than 200 nucleotides (e.g., about 200 to about 1200 nt, about 2500 nt or more) that lack an extended open reading frame (ORF). The term "non-coding RNA" (ncRNA) includes lncRNA as well as shorter transcripts, such as transcripts less than about 200 nt, such as about 30 to 200 nt.

[0116] Therefore, in some embodiments, the delivery of ncRNA, such as delivery to a specific brain structure of interest, corrects abnormal RNA expression levels or regulates the level of pathogenic lncRNA. Therefore, in some embodiments, the present invention provides rAAV, wherein the viral genome is modified to encode therapeutic non-coding RNA (ncRNA). In some embodiments, ncRNA is a long non-coding RNA (lncRNA) of about 200 nucleotides (nucleotide, nt) or longer. In some embodiments, the therapeutic agent is an ncRNA of about 25nt or about 30nt to about 200nt in length. In some embodiments, the length of lncRNA is about 200nt to about 1,200nt. In some embodiments, the length of lncRNA is about 200nt to about 1,100, about 1,000, about 900, about 800, about 700, about 600, about 500, about 400 or about 300nt.

[0117] C. CRISPR system

[0118] Gene editing is a technology that allows the modification of targeted genes within living cells. Recently, the bacterial immune system using CRISPR to perform gene editing on demand has revolutionized the way scientists perform genome editing. The Cas9 protein of the CRISPR system is an RNA-guided DNA endonuclease that can be relatively easily engineered to target new sites by changing the sequence of its guide RNA. This discovery makes sequence-specific gene editing functionally effective.

[0119] In general, a "CRISPR system" collectively refers to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (encompassing "direct repeats", as well as partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from a CRISPR locus.

[0120] CRISPR / Cas nuclease or CRISPR / Cas nuclease system can comprise non-coding RNA molecule (guide) RNA, which binds specifically to DNA sequence; and Cas protein (e.g., Cas9), which has nuclease functionality (e.g., two nuclease domains). One or more elements of CRISPR system can be derived from type I, type II or type III CRISPR system, for example, from a specific organism comprising endogenous CRISPR system, such as Streptococcus pyogenes.

[0121] The CRISPR system can induce double strand breaks (DSBs) at the target site, followed by destruction as discussed herein. In other embodiments, Cas9 variants considered to be "nicking enzymes" are used to nick a single strand at the target site. Paired nicking enzymes can be used, for example, to improve specificity, each enzyme being guided by a different pair of gRNA targeting sequences, so that when the nicks are introduced at the same time, 5' overhangs are introduced. In other embodiments, the catalytically inactive Cas9 is fused to a heterologous effector domain (e.g., a transcriptional repressor (e.g., KRAB) or an activator) to affect gene expression. Alternatively, the CRISPR system with a catalytically inactive Cas9 further comprises a transcriptional repressor or activator fused to a ribosome binding protein.

[0122] In some aspects, Cas nuclease and gRNA (including a fusion of crRNA and fixed tracrRNA specific to the target sequence) are introduced into the cell. In general, the target site at the 5' end of 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 the 5' position of its adjacent protospacer adjacent motif (protospacer adjacent motif, PAM) sequence (e.g., usually NGG or NAG) next to the original spacer. In this aspect, 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. In general, the CRISPR system is characterized by promoting the elements of the formation of CRISPR complexes at the target sequence site. Generally speaking, "target sequence" generally refers to a sequence designed to have complementarity in which the hybridization between the target sequence and the guide sequence promotes the formation of CRISPR complexes. Perfect complementarity is not necessarily required if there is sufficient complementarity to cause hybridization and promote formation of the CRISPR complex.

[0123] The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, such as within an organelle of a cell. In general, a sequence or template that can be used to reorganize a target locus comprising a target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, recombination is homologous recombination.

[0124] Generally speaking, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence). A tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracr sequence), can also form part of a CRISPR complex, for example by hybridizing along at least a portion of the tracr sequence to all or a portion of a tracr partner sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr partner sequence to hybridize and participate in the formation of the CRISPR complex, e.g., at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr partner sequence when optimally aligned.

[0125] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into the cell so that the expression of the elements of the CRISPR system guides the formation of the CRISPR complex at one or more target sites. The components can also be delivered to the cell as proteins and / or RNA. For example, the Cas enzyme, the guide sequence connected to the tracr partner sequence, and the tracr sequence can each be operably connected to a separate regulatory element on a separate vector. The Cas enzyme can be a target gene under the control of a regulated alternative splicing event, as disclosed herein, as a chimeric target gene minigene or as a target gene for a chimeric minigene transactivator. The gRNA can be under the control of a constitutive promoter.

[0126] Alternatively, two or more elements expressed by the same or different regulatory elements can be combined in a single vector, while one or more additional vectors provide any components of the CRISPR system not contained in the first vector. The vector may contain 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. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in the cell.

[0127] The vector may comprise a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme (e.g., a Cas protein). Some non-limiting examples of Cas proteins include

[0128] 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, Csxl, Csx15, Csfl, Csf2, Csf3, Csf4, homologs thereof or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0129] The CRISPR enzyme can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae (S.pneumonia)). The CRISPR enzyme can guide the cutting of one or both chains at the location of the target sequence, such as within the target sequence and / or within the complementary sequence of the target sequence. The vector can encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme, so that the mutated CRISPR enzyme lacks the ability to cut one or both chains of the target polynucleotide containing the target sequence. For example, the substitution of aspartic acid to alanine (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cuts two chains to a nickase (cutting a single strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence (e.g., two guide sequences) that target the sense and antisense strands of a DNA target, respectively. This combination allows the two chains to be nicked and used to induce NHEJ or HDR.

[0130] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in a specific cell, such as a eukaryotic cell. Eukaryotic cells can be those of a specific organism or derived from a specific organism, such as a mammal, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a target host cell by replacing at least one codon of a native sequence with a more frequent or most frequently used codon in the genes of the target host cell while maintaining the native amino acid sequence. Various species show specific preferences for certain codons of specific amino acids. Codon bias (differences in codon usage between organisms) is generally associated with the translation efficiency of messenger RNA (mRNA), which in turn is believed to depend on, among other things, the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The advantage of the selected tRNA in the cell generally reflects the most frequently used codons in peptide synthesis. Therefore, genes can be customized based on codon optimization for optimal gene expression in a given organism.

[0131] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0132] Optimal alignment can be determined using any suitable algorithm for aligning sequences, some non-limiting examples of which include the Smith-Waterman algorithm; the Needleman-Wunsch algorithm; algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner); Clustal W; Clustal X; BLAT; Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.)); SOAP (available at soap.genomics.org.cn) and Maq (available at maq.sourceforge.net).

[0133] The CRISPR enzyme can be part of a fusion protein comprising one or more heterologous protein domains. The CRISPR enzyme fusion protein may include any additional protein sequence, and optionally a linker sequence between any two domains. Some 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, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Some non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (influenza hemagglutinin, HA) tags, Myc tags, VSV-G tags, and thioredoxin (thioredoxin, Trx) tags. Some examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins, including blue fluorescent protein (BFP). The CRISPR enzyme can be fused to a gene sequence encoding a protein or protein fragment that binds to a DNA molecule or binds to other cellular molecules, including but not limited to maltose binding protein (MBP), S tag, Lex A DNA binding domain (DBD) fusion, GAL4A DNA binding domain fusion, and herpes simplex virus (HSV) BP16 protein fusion. Additional domains that can form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, which is incorporated herein by reference.

[0134] D. Therapeutic proteins

[0135] Some embodiments relate to the expression of recombinant proteins and polypeptides. Such proteins can be neuroprotective proteins, such as anti-apoptotic proteins, antioxidant enzymes (e.g., those belonging to the superoxide dismutase (SOD) family), neurotrophic factors / neuroprotective factors, or anti-inflammatory proteins. The therapeutic protein can be Birc1a (NAIP), Birc2 (c-IAP1 / HIAP-2), Birc3 (cIAP-2 / HIAP-1), Birc4 (XIAP), Birc5 (survivin), Birc6 (apollon), Birc7 (livin), Birc8 (TsIAP); members of the Bcl-2 family: Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bcl-2L10, BFL-1; c-Jun N-terminus kinase (c-Jun N-terminus kinase) known as Jun interacting protein (JIP) (JIP-1, JIP-2, JIP-3, JIP-4) endogenous inhibitors of JNK; SOD1, SOD2; catalase; peroxiredoxin-1, peroxiredoxin-2, glutathione preoxidase 1 (Gpx1), Gpx2, Gpx3, or Gpx4; NGF, BDNF, CNTF, GDNF, growth / differentiation factor-15 (GDF-15), erythropoietin, or vascular endothelial growth factor (VEGF); interleukin-10 (IL-10); glutathione S-transferase, annexin-1 (ANXA1), inhibitor of NF-κB NF-κB, IκB); USH1, USH1G (also known as SANS), CIB2 (calcium and integrin binding protein 2), ATOH-1, ACTG1, ATP2B2, CDH23 (cadherin 23),

[0136] CLDN14, CLRN1, CoCH, COL11A2, DFNA5, DFNB31, DFNB59, ESPN, EYA4, GJB2, GJB3, GJB6, KCNQ4, LHFPL5, MT-RNR1, MT-TS1, MYO1A, MYO6, MYO7A (Myosin 7a), MYO15A, OTOF, PCDH15 (Protocadherin 15), PDZD7, POU3F4, SLC26A4, STRC, TECTA, TMC1, TMC2, TMIE, TMPRSS3, TRI0BP, USH1C, VLGR1, WFS1, ACTG1, ADCY1, ATOFf1, ATP6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD164, CDC 14 A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A 1, COL9A2, COL1 1 A1, COL1 1A2, CRYM, DCDC2, DFNA5, DFNB31, DFNB59, DIAPH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, MET, MIR183, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MY015A, MYOIA, MY03A, MY06, MY07A, NARS2, NDP, NF2, NT3, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, SMAC / DIABLO, SNAI2, SOX10,STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGR1, WFS1, WHRN or XIAP. ,

[0137] When the present application refers to the function or activity of a "modified protein" or "modified polypeptide", one of ordinary skill in the art will understand that this includes, for example, a protein or polypeptide that has additional advantages relative to an unmodified protein or polypeptide. It is specifically contemplated that embodiments regarding a "modified protein" may be implemented relative to a "modified polypeptide", and vice versa.

[0138] Recombinant proteins may have a deletion and / or replacement of amino acids; therefore, proteins with deletions, proteins with replacements, and proteins with deletions and replacements are modified proteins. In some embodiments, these proteins may also include inserts or added amino acids, such as proteins with fusion proteins or with joints. "Modified deletion proteins" lack one or more residues of native proteins, but may have the specificity and / or activity of native proteins. "Modified deletion proteins" may also have reduced immunogenicity or antigenicity. An example of a modified deletion protein is a protein with amino acid residues missing from at least one antigenic region (i.e., a region of a protein determined to be antigenic in a specific organism (e.g., an organism to which the modified protein is administered)).

[0139] Replacement or substitution variants generally include an exchange of one amino acid with another amino acid at one or more sites within a protein, and can be designed to modulate one or more properties of a polypeptide, particularly its effector function and / or bioavailability. Replacement may or may not be conservative, i.e., one amino acid is replaced by one of similar shape and charge. Conservative replacements are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0140] In addition to deletions or substitutions, modified proteins may have insertions of residues, which typically involve adding at least one residue in a polypeptide. This may include insertions of targeting peptides or polypeptides or only single residues. Terminal additions, referred to as fusion proteins, are discussed below.

[0141] The term "biological functional equivalent" is well known in the art and is further defined in detail 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 the control polypeptide are included, provided that the biological activity of the protein is maintained. In certain aspects, the recombinant protein and its natural counterpart can be biologically functionally equivalent.

[0142] It will also be understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, and still be substantially as shown in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including, where protein expression is concerned, maintaining biological protein activity. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may, for example, contain various non-coding sequences flanking the 5' or 3' portion of the coding region, or may contain various internal sequences known to exist within genes, i.e., introns.

[0143] Protein or peptide used herein generally refers to, but is not limited to, proteins greater than about 200 amino acids to the full-length sequence translated from a gene; polypeptides greater than about 100 amino acids; and / or peptides of about 3 to about 100 amino acids. For convenience, the terms "protein", "polypeptide" and "peptide" are used interchangeably herein.

[0144] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimetic known in the art. In certain embodiments, the residues of a protein or peptide are continuous, without any non-amino acid interrupting the sequence of amino acid residues. In other embodiments, the sequence may contain one or more non-amino acid moieties. In some specific embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0145] Thus, the term "protein or peptide" encompasses an amino acid sequence comprising at least one of the 20 common amino acids found in naturally occurring proteins or at least one modified or unusual amino acid.

[0146] Certain embodiments of the present invention relate to fusion proteins. These molecules may have therapeutic proteins connected to heterologous domains at the N- or C-terminal ends. For example, fusions may also use leader sequences from other species to allow recombinant expression of proteins in heterologous hosts. Another useful fusion includes adding protein affinity tags, for example, serum albumin affinity tags or six histidine residues, or immunologically active domains, such as antibody epitopes, preferably cleavable, to facilitate purification of fusion proteins. Non-limiting affinity tags include polyhistidine, chitin binding protein (chitin binding protein, CBP), maltose binding protein (MBP) and glutathione-S-transferase (GST).

[0147] Methods for producing fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by synthesizing the complete fusion protein de novo, or by attaching a DNA sequence encoding a heterologous domain, followed by expressing the complete fusion protein.

[0148] By connecting the gene with a bridging DNA fragment encoding a peptide linker (splicing between the tandemly linked polypeptides), the production of a fusion protein that restores the functional activity of the parent protein can be facilitated. The linker will be of sufficient length to allow the correct folding of the resulting fusion protein.

[0149] The expression of the transgene can be directed by the natural promoter of the transgene (i.e., a naturally occurring promoter with a transgene coding sequence) or the expression of the transgene can be directed by a heterologous promoter (e.g., CMV promoter, Espin promoter, PCDH15 promoter, PTPRQ promoter, and TMHS (LHFPL5) promoter). For example, any transgene described herein can be used with its natural promoter. Alternatively, any transgene described herein can be used with a heterologous promoter. The heterologous promoter used herein refers to a promoter that does not naturally direct the expression of the sequence (i.e., the sequence is not found in nature). Representative heterologous promoters that can be used to direct the expression of any transgene indicated herein include, for example, CMV promoter, CBA promoter, CASI promoter, P promoter, and EF-1 promoter, α9 nicotinic receptor promoter, prestin promoter, Gfil promoter, and Vglut3 promoter. In addition, a promoter that naturally directs the expression of one of the transgenes mentioned above (e.g., KCNQ4 promoter, Myo7a promoter, Myo6 promoter, or Atohl promoter) can be used as a heterologous promoter to direct the expression of the transgene. In other embodiments, the promoter is an Espin promoter, a PCDH15 promoter, a PTPRQ promoter, and a TMHS (LHFPL5) promoter.

[0150] III. Methods of Treatment and Administration

[0151] In some aspects, viral vectors can be directly applied to patients (in vivo), or they can be used to treat cells in vitro or ex vivo and then administered to patients. The term "vector" refers to a small carrier nucleic acid molecule, plasmid, virus (e.g., AAV vector, retroviral vector, lentiviral vector), or other carrier that can be manipulated by inserting or incorporating nucleic acid. Vectors (e.g., viral vectors) can be used to introduce / transfer nucleic acid sequences into cells so that the nucleic acid sequences therein are transcribed and, if encoding proteins, are subsequently translated by cells.

[0152] Any suitable cell or mammal can be applied or treated by the methods or uses described herein. Generally speaking, the mammals that need the methods described herein are suspected of having or expressing abnormal or abnormal proteins associated with disease states. Alternatively, the mammal recipient may suffer from a disease suitable for gene replacement therapy. "Gene replacement therapy" used herein refers to the administration of exogenous genetic material encoding therapeutic agents to the recipient and the subsequent in situ expression of the administered genetic material. Therefore, the phrase "a disease suitable for gene replacement therapy" covers such diseases, such as hereditary diseases (i.e., disease conditions attributed to one or more gene defects), acquired conditions (i.e., pathological conditions not attributed to congenital defects), cancer and preventive processes (i.e., prevention of diseases or undesirable medical conditions). Therefore, the term "therapeutic agent" used herein refers to any agent or substance that has a beneficial effect on the mammal recipient. Therefore, "therapeutic agent" covers both therapeutic molecules and preventive molecules with nucleic acid or protein components.

[0153] Some non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cattle, goats, sheep, pigs), and experimental animals (e.g., mice, rats, rabbits, guinea pigs). In certain embodiments, the mammal is a human. In certain embodiments, the mammal is a non-rodent mammal (e.g., a human, pig, goat, sheep, horse, dog, etc.). In certain embodiments, the non-rodent mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult, teenager, child, infant, or mammal in utero). The mammal can be male or female. In certain embodiments, the mammal can be an animal disease model, for example, an animal model having or expressing an abnormal or abnormal protein associated with a disease state, or an animal model that does not adequately express a protein and causes a disease state.

[0154] Mammals (subjects) treated by the methods or compositions described herein include adults (18 years or older) and children (less than 18 years old). Adults include elderly people. Representative adults are 50 years or older. Children are 1 to 2 years old, or 2 to 4, 4 to 6, 6 to 18, 8 to 10, 10 to 12, 12 to 15, and 15 to 18 years old. Children also include infants. Infants are generally 1 to 12 months old.

[0155] In certain embodiments, the method comprises administering a plurality of viral particles to a mammal as described herein, wherein the severity, frequency, progression or onset of one or more symptoms of a disease state (e.g., a neurodegenerative disease) is reduced, lowered, blocked, inhibited or delayed. In certain embodiments, the method comprises administering a plurality of viral particles to a mammal to treat adverse symptoms of a disease state (e.g., a neurodegenerative disease). In certain embodiments, the method comprises administering a plurality of viral particles to a mammal to stabilize, delay or prevent the deterioration or progression of adverse symptoms of a disease state (e.g., a neurodegenerative disease), or to reverse adverse symptoms of a disease state (e.g., a neurodegenerative disease).

[0156] In certain embodiments, the method comprises administering a plurality of viral particles to the central nervous system, or portion thereof, of a mammal as described herein, and the severity, frequency, progression, or onset of one or more symptoms of a disease state (e.g., a neurodegenerative disease) is reduced, decreased, arrested, inhibited, or delayed 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.

[0157] In certain embodiments, symptoms or adverse effects comprise early, intermediate or late symptoms; behavioral, personality or language symptoms; swallowing, movement, seizure, tremor or restlessness symptoms; ataxia; and / or cognitive symptoms such as memory, organizational skills.

[0158] IV. Pharmaceutical Compositions

[0159] The terms "pharmaceutically acceptable" and "physiologically acceptable" as used herein mean a biologically acceptable composition, formulation, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, routes of delivery in vivo, or routes of contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is not a biologically or otherwise undesirable substance, for example, the substance can be administered to a subject without causing substantial undesirable biological effects. Such compositions, "pharmaceutically acceptable" and "physiologically acceptable" formulations and compositions can be sterile. Such pharmaceutical preparations and compositions can be used, for example, to administer viral particles to a subject.

[0160] Such formulations and compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic agents, and absorption enhancers or absorption delaying agents that are compatible with drug administration or in vivo contact or in vivo delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents. Supplementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents, and antifungal agents) may also be incorporated into the formulations and compositions.

[0161] Pharmaceutical compositions generally include pharmaceutically acceptable excipients. Such excipients include any agent that does not induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may be included therein, for example: inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances such as surfactants, wetting agents or emulsifiers, pH buffer substances, etc. may be present in such carriers.

[0162] The pharmaceutical composition can be formulated to be compatible with a particular route of administration or delivery as indicated herein or known to those skilled in the art. Thus, the pharmaceutical composition comprises a carrier, diluent or excipient suitable for administration or delivery by a variety of routes.

[0163] The pharmaceutical form suitable for injection or infusion of viral particles may include sterile aqueous solution or dispersion, which is suitable for the temporary preparation of sterile injectable or infusible solution or dispersion optionally encapsulated in liposome. In all cases, the final form should be sterile fluid and stable under preparation, use and storage conditions. Liquid carrier or carrier can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oil, non-toxic glyceride and its suitable mixture. Suitable fluidity can be, for example, by forming liposome, by maintaining the required particle size in the case of dispersion or by using a surfactant to maintain. Isotonic agents may be included, such as sugar, buffer or salt (for example, sodium chloride). The extended absorption of injectable compositions can be achieved by using delayed absorption agents such as aluminum monostearate and gelatin in the compositions.

[0164] The solution or suspension of viral particles may optionally include one or more of the following components: a sterile diluent such as water for injection, saline solution (e.g., phosphate buffered saline (PBS)), artificial CSF, a surfactant, a fixed oil, a polyol (e.g., 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.; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose.

[0165] Pharmaceutical formulations, compositions and delivery systems suitable for the compositions, methods and uses of the present invention are known in the art (see, e.g.,

[0166] Remington: The Science and Practice ofPharmacy(2003)20 th ed., Mack Publishing Co, Easton, PA; Remington's Pharmaceutical Sciences (1990) 18 th ed., MackPublishing Co., Easton, PA; The Merck Index (1996)12 th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), TechnonicPublishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11 th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), RL Juliano, ed., Oxford, NY, pp. 253-315).

[0167] For ease of administration and uniformity of dosage, the viral particles and compositions thereof can be formulated in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as single doses for individuals to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The dosage unit form depends on the number of viral particles considered necessary to produce the desired effect. The necessary amount can be formulated in a single dose, or it can be formulated in multiple dose units. The dose can be adjusted to an appropriate viral particle concentration, optionally combined with an anti-inflammatory agent, and packaged for use.

[0168] In one embodiment, the pharmaceutical composition will contain sufficient genetic material to provide a therapeutically effective amount, ie, an amount sufficient to alleviate or ameliorate the symptoms or adverse effects of the disease state in question or an amount sufficient to confer a desired benefit.

[0169] As used herein, "unit dosage form" refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit containing a predetermined amount is optionally associated with a pharmaceutical carrier (excipient, diluent, carrier or filler), which is calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect) when the pharmaceutical carrier is administered in one or more doses. The unit dosage form may be in ampoules and vials, for example, which may contain a liquid composition or a composition in a freeze-dried or lyophilized state; for example, a sterile liquid carrier may be added prior to in vivo administration or delivery. A single unit dosage form may be contained in a multi-dose kit or container. Thus, for ease of administration and uniformity of dosage, for example, viral particles and pharmaceutical compositions thereof may be packaged in single or multiple unit dosage forms.

[0170] The formulation comprising the viral particles generally comprises an effective amount, which is readily determined by one skilled in the art. The viral particles may generally be from about 1% to about 95% (w / w) of the composition, or even higher if appropriate. The amount to be administered depends on factors such as the age, weight and physical condition of the mammal or human subject being treated. The effective dose may be determined by one of ordinary skill in the art by routine experiments establishing a dose response curve.

[0171] V. Definition

[0172] The terms "polynucleotide", "nucleic acid" and "transgenic" are used interchangeably herein to refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and polymers thereof. Polynucleotides include genomic DNA, cDNA and antisense DNA, as well as spliced ​​or unspliced ​​mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, such as small or short hairpin (sh) RNA, micro RNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA or antisense RNA). Polynucleotides may include naturally occurring, synthetic and intentionally modified or altered polynucleotides (e.g., variant nucleic acids). Polynucleotides may be single-stranded, double-stranded or triple-stranded, linear or circular, and may have any suitable length. When discussing polynucleotides, the sequence or structure of a specific polynucleotide may be described herein according to the convention of providing a sequence in the 5' to 3' direction.

[0173] A nucleic acid encoding a polypeptide generally comprises an open reading frame encoding the polypeptide. Unless otherwise indicated, a specific nucleic acid sequence also includes degenerate codon substitutions.

[0174] The nucleic acid may comprise one or more expression control or regulatory elements operably linked to the open reading frame, wherein the one or more regulatory elements are configured to direct the transcription and translation of the polypeptide encoded by the open reading frame in mammalian cells. Some 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, poly A sequences, secretion sequences, etc. Expression control / regulatory elements may be obtained from the genome of any suitable organism.

[0175] "Promoter" refers to a nucleotide sequence that is usually located upstream (5') of a coding sequence and directs and / or controls the expression of a coding sequence by providing recognition for RNA polymerase and other factors required for proper transcription. Pol II promoters contain a minimal promoter, which is a short DNA sequence containing a TATA box and optionally other sequences for specifying a transcription start site, to which regulatory elements are added to control expression. Type 1 pol III promoters contain three cis-acting sequence elements downstream of the transcription start site: a) 5' sequence element (A block); b) middle sequence element (I block); c) 3' sequence element (C block). Type 2 pol III promoters contain two essential cis-acting sequence elements downstream of the transcription start site: a) A box (5' sequence element); and b) B box (3' sequence element). Type 3 pol III promoters contain several cis-acting promoter elements upstream of the transcription start site, such as the traditional TATA box, proximal sequence element (PSE) and distal sequence element (DSE).

[0176] An "enhancer" is a DNA sequence that stimulates transcriptional activity and can be an intrinsic element of a promoter or a heterologous element that enhances expression levels or tissue specificity. It can operate in either direction (5'->3' or 3'->5') and can function even if located upstream or downstream of a promoter.

[0177] Promoters and / or enhancers may be derived entirely from a native gene, or may be composed of different elements derived from different elements found in nature, or may even comprise synthetic DNA segments. A promoter or enhancer may comprise a DNA sequence that participates in the binding of protein factors that regulate / control the effectiveness of transcription initiation in response to a stimulus, physiological or developmental condition.

[0178] Some non-limiting examples of promoters include SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, pol II promoter, pol III promoter, synthetic promoter, hybrid promoter, etc. In addition, sequences derived from non-viral genes such as mouse metallothionein gene will also be used herein. Some exemplary constitutive promoters include promoters for the following genes encoding certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, actin promoter, U6, and other constitutive promoters known to those skilled in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include: the early and late promoters of SV40; the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses; and the thymidine kinase promoter of herpes simplex virus, etc. Therefore, any of the constitutive promoters mentioned above can be used to control the transcription of heterologous gene inserts.

[0179] As used herein, "transgene" conveniently refers to a nucleic acid sequence / polynucleotide that is intended to be or has been introduced into a cell or organism. A transgene includes any nucleic acid, such as a gene encoding an inhibitory RNA or a polypeptide or protein, and is generally heterologous to the naturally occurring AAV genomic sequence.

[0180] The term "transduction" refers to the introduction of a nucleic acid sequence into a cell or host organism by means of a vector (e.g., a viral particle). Therefore, the introduction of a transgene into a cell by a viral particle can be referred to as the "transduction" of a cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. If the introduced transgene is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in the cell or organism, and further transmitted to the daughter cells or organism of the recipient cell or organism or inherited by it. Finally, the introduced transgene may be present outside the chromosome of the recipient cell or host organism, or only exist temporarily. Therefore, "transduced cells" are cells into which a transgene is introduced by means of transduction. Therefore, "transduced" cells are cells or their progeny into which a transgene has been introduced. Transduced cells can proliferate, transcribe transgenes and express encoded inhibitory RNA or protein. For gene therapy purposes and methods, transduced cells can be in mammals.

[0181] Transgenics under the control of an inducible promoter are expressed only or to a greater extent in the presence of an inducer (e.g., transcription under the control of a metallothionein promoter is greatly increased in the presence of certain metal ions). Inducible promoters include responsive elements (REs) that stimulate transcription when combined with their inducing factors. For example, there are REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. Promoters containing specific REs can be selected to obtain inducible responses, and in some cases, the REs themselves can be connected to different promoters to confer inducibility to the recombinant gene. Therefore, by selecting a suitable promoter (constitutive vs inducible; strong vs weak), both the presence and expression level of the polypeptide in genetically modified cells can be controlled. If the gene encoding the polypeptide is under the control of an inducible promoter, the in situ delivery of the polypeptide is triggered by exposing the genetically modified cells in situ to conditions that allow transcription of the polypeptide, for example, by intraperitoneal injection of a specific inducer of the inducible promoter that controls the transcription of the agent. For example, in situ expression by a genetically modified cell of a polypeptide encoded by a gene under the control of a metallothionein promoter is enhanced by contacting the genetically modified cell in situ with a solution containing the appropriate (ie, inducing) metal ions.

[0182] A nucleic acid / transgene is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. A nucleic acid / transgene encoding an RNAi or polypeptide or a nucleic acid directing expression of a polypeptide may contain an inducible promoter or a tissue-specific promoter for controlling transcription of the encoded polypeptide. A nucleic acid operably linked to an expression control element may also be referred to as an expression cassette.

[0183] In certain embodiments, cell type specific promoters or inducible promoters, enhancers, etc. are used in the methods and uses described herein. Some non-limiting examples of cell type specific promoters include those isolated from genes from TMC1, TMC2, Espin, PCDH15, PTPRQ, TMHS (LHFPL5), MYO1A. Some non-limiting examples of inducible promoters include DNA responsive elements for ecdysone, tetracycline, hypoxia, and IFN.

[0184] 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.

[0185] As used herein, the terms "modification" or "variant" and grammatical variations thereof refer to deviations of a nucleic acid, polypeptide, or subsequence thereof from a reference sequence. Thus, modified and variant sequences may have substantially the same, higher or lower expression, activity, or function as a reference sequence, but retain at least some of the activity or function of the reference sequence. A specific type of variant is a mutant protein, which refers to a protein encoded by a gene having a mutation, such as a missense or nonsense mutation.

[0186] A "nucleic acid" or "polynucleotide" variant refers to a modified sequence that has been genetically altered compared to the wild type. A sequence can be genetically modified without changing the encoded protein sequence. Alternatively, a sequence can be genetically modified to encode a variant protein. A nucleic acid or polynucleotide variant can also refer to a combined sequence that has been codon-modified to encode a protein that still retains at least partial sequence identity with a reference sequence (e.g., a wild-type protein sequence) and has also been codon-modified to encode a variant protein. For example, some codons of such a nucleic acid variant will be changed without changing the amino acids of the protein encoded thereby, and some codons of the nucleic acid variant will be changed, which in turn changes the amino acids of the protein encoded thereby.

[0187] The terms "protein" and "polypeptide" are used interchangeably herein. "Polypeptides" encoded by "nucleic acids" or "polynucleotides" or "transgenes" disclosed herein include partial or full-length native sequences, such as naturally occurring wild-type and functional polymorphic proteins, functional subsequences (fragments), and sequence variants thereof, as long as the polypeptide retains a certain degree of function or activity. Therefore, in the methods and uses of the present invention, such polypeptides encoded by nucleic acid sequences do not need to be identical to endogenous proteins that are defective or whose activity, function or expression is insufficient, missing or absent in the treated mammal.

[0188] Some non-limiting examples of modifications include one or more nucleotide or amino acid replacements (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).

[0189] An example of an amino acid modification is a conservative amino acid substitution or deletion.In some embodiments, the modified sequence or variant sequence retains at least part of the function or activity of the unmodified sequence (eg, wild-type sequence).

[0190] Another example of amino acid modification is the introduction of targeting peptides into the capsid protein of the viral particle.Peptides have been identified that target recombinant viral vectors to the central nervous system, such as different brain regions.

[0191] The recombinant virus thus modified may preferentially bind to one type of tissue (e.g., CNS tissue) rather than another type of tissue (e.g., liver tissue). In certain embodiments, a recombinant virus carrying a modified capsid protein may "target" cerebrovascular epithelial tissue by binding at a level higher than that of a comparable unmodified capsid protein. For example, a recombinant virus having a modified capsid protein may bind to cerebrovascular epithelial tissue at a level 50% to 100% higher than that of an unmodified recombinant virus.

[0192] "Nucleic acid fragment" is a part of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in most organisms is genetic material, and ribonucleic acid (RNA) is involved in transferring the information contained in DNA to proteins. Fragments and variants of the disclosed nucleotide sequences and proteins or partial length proteins encoded therefrom are also encompassed in the present invention. "Fragment" or "part" means the full length or less than the full length of the nucleotide sequence or amino acid sequence encoding a polypeptide or protein. In certain embodiments, a fragment or part is biologically functional (i.e., retaining 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the activity or function of the wild type).

[0193] A "variant" of a molecule is a sequence that is substantially similar to the sequence of a natural molecule. For nucleotide sequences, variants include those sequences that encode the same amino acid sequence as the natural protein due to the degeneracy of the genetic code. For example, these naturally occurring allelic variants can be determined using molecular biology techniques (such as, for example, using polymerase chain reaction (PCR) and hybridization techniques). Variant nucleotide sequences also include nucleotide sequences of synthetic origin, such as those produced by using site-directed mutagenesis (which encodes natural proteins), and those encoding polypeptides with amino acid substitutions. In general, the nucleotide sequence variants of the present invention will have at least 40%, 50%, 60% to 70%, for example 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% to 79%, usually at least 80%, for example 81% to 84%, at least 85%, for example 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% to 98% sequence identity with 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 activity or function of the wild-type).

[0194] "Conservative variation" of a particular nucleic acid sequence refers to those nucleic acid sequences that encode the same or substantially the same amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For example, codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Therefore, at each position where arginine is specified by a codon, the codon can be changed to any corresponding codon described without changing the encoded protein. Such nucleic acid variation is a "silent variation", which is a "conservatively modified variation". Unless otherwise indicated, each nucleic acid sequence encoding a polypeptide described herein also describes each possible silent variation. Those skilled in the art will recognize that each codon in a nucleic acid (except ATG, which is typically the only codon for methionine) can be modified to produce functionally identical molecules by standard techniques. Therefore, each "silent variation" of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0195] The term "significant identity" of a polynucleotide sequence means that the polynucleotide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, or at least 90%, 91%, 92%, 93% or 94%, or even at least 95%, 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using one of the alignment programs using standard parameters. One skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc. Significant identity of amino acid sequences for these purposes generally means at least 70%, at least 80%, 90% or even at least 95% sequence identity.

[0196] In the case of polypeptides, the term "substantially identical" means that the polypeptide comprises a sequence having 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 the reference sequence over a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunoreactive with antibodies raised against a second polypeptide. Thus, for example, a polypeptide is identical to a second polypeptide where the two peptides differ only in conservative substitutions.

[0197] "Mechanosensation" means a response to a mechanical stimulus. Touch, hearing, and balance are examples of the conversion of mechanical stimuli into neuronal signals. The conversion of mechanosensory input into a response to a mechanical stimulus is through a process known as "mechanotransduction."

[0198] "Disease" means any disorder or obstacle that damages or interferes with the normal function of a cell, tissue or organ. Some examples of disease include genetic disorders, characterized in that the function of a protein that functions in mechanical sensory transduction is lost, such as expressed in the inner ear of a subject. In another embodiment, the disease is Usher Syndrome (UsherSyndrome) (e.g., USH1) or age-related hearing loss. In one embodiment, the disease is hearing impairment associated with a genetic defect, such as a defect in TMC1, TMC2, MY07A, USCH1 C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHKN, CLRN1, PDZD7, USH1C (e.g., harmonin-a, b or c).

[0199] The term "treatment" and its variations refer to both therapeutic treatment and preventive or preventive measures, wherein the purpose is to prevent, inhibit, alleviate or reduce undesirable physiological changes or disorders, such as the occurrence, progression or deterioration of disorders. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, weakening of disease extent, stabilization of symptoms or adverse effects of the disease (i.e., no deterioration or progression), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonged survival compared to the expected survival without treatment. Those in need of treatment include those already suffering from the disease or disorder and those susceptible to the disease (e.g., as determined by genetic assays).

[0200] As used herein, "substantially free" with respect to a specified component is used herein to mean that none of the specified component is purposefully formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified component resulting from any accidental contamination of the composition is much less than 0.05%, preferably less than 0.01%. Most preferred are compositions wherein the amount of the specified component cannot be detected by standard analytical methods.

[0201] As used herein in the specification, a noun without a quantifier may mean one or more. As used herein in the claims, when used in conjunction with the word "comprises", a noun without a quantifier may mean one or more than one.

[0202] Unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, the use of the term "or / or" in the claims is used to mean "and / or / or", but the present disclosure supports a definition referring to only alternatives and "and / or / or". As used herein, "another" may mean at least a second or more.

[0203] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation in error for the device being employed to determine the value, the inherent variation in the method being employed to determine the value, the variation that exists between study subjects, or a value within 10% of the stated value.

[0204] VI.Medicine Box

[0205] The invention provides a kit having packaging material and having one or more components therein. The kit typically comprises a label or package insert containing a description of the components or instructions for use of the components therein in vitro, in vivo or ex vivo. The kit may comprise a collection of such components (e.g., nucleic acids, recombinant vectors, and / or viral particles).

[0206] A kit refers to a physical structure that houses one or more components of the kit. The packaging material can maintain the components sterile and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0207] The label or insert may include information on one or more of the components, dosage amounts, clinical pharmacology (including mechanism of action, pharmacokinetics and pharmacodynamics) of the active ingredient. The label or insert may include information indicating the manufacturer, batch number, place of production and date of production, and expiration date. The label or insert may include information indicating manufacturer information, batch number, manufacturer location and date. The label or insert may include disease information for which the kit components can be used. The label or insert may include instructions for clinicians or subjects to use one or more of the kit components in methods, uses, treatment schemes or therapeutic regimens. The instructions may include dosage amounts, frequency or duration, and instructions for implementing any method, use, treatment scheme or prevention or therapeutic regimen described herein.

[0208] The label or insert may contain information about any benefits (e.g., preventive or therapeutic benefits) that the components may provide. The label or insert may contain information about potential adverse side effects, complications, or reactions, such as warning the subject or clinician about situations in which the particular composition is not suitable for use. Adverse side effects or complications may also occur when the subject has, will, or is taking one or more other drugs, which may be incompatible with the composition or with another treatment regimen or therapeutic regimen that the subject has, will, or is experiencing that is incompatible with the composition, and therefore, the instructions may contain information about such incompatibilities.

[0209] Labels or inserts include "printed matter", such as paper or paperboard, either alone or fixed to a component, a kit or packaging material (e.g., a box), or attached to an ampoule, tube or vial containing a kit component. Labels or inserts may additionally include computer-readable media, such as barcode printed labels, disks, optical disks (e.g., CD- or DVD-ROM / RAM, DVD), MP3, or electronic storage media (e.g., RAM and ROM) or a mixture of these such as magnetic / optical storage media, flash memory, hybrids, and memory type cards.

[0210] VII. Examples

[0211] The following examples are included to illustrate some preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for the practice of the present invention. However, it will be appreciated by those skilled in the art based on this disclosure that many changes may be made to some of the disclosed specific embodiments and still the same or similar results may be obtained without departing from the spirit and scope of the present invention.

[0212] Example 1 - Therapeutic Delivery to the Globus Pallidus

[0213] Libraries of peptide-modified AAV capsid variants were generated by inserting random targeting peptide sequences into AAV1, AAV2, and AAV9 at position 590, 587, and 588 of the AAV1, AAV2, and AAV9 capsids, respectively. These libraries were screened in rhesus monkeys for variants that broadly transduced deep brain structures and the cerebral cortex after infusion into the globus pallidus. Figures 38A to 38C Six variants were selected for further validation in fluorescence experiments in two additional rhesus monkeys (Table 1). At least five capsid variants transduced deep brain structures affected in HD and PD. Transduction by AAV1.THTD.mTFP1 was extensive in the caudate / putamen. AAV1.THTD.mTFP1 also transduced a large number of layer V / VI cortical projection neurons ( Fig.39 These AAV capsids can be used to package a variety of therapeutic payloads for HD and PD. Liver transduction of these variants was below the limit of detection ( Fig.40 )(AAV-BLD010 is AAV1.THTD.mTFP1; AAV-BLD011 is AAV2.SGGR.NES_mNG; AAV-BLD012 is AAV2.SKGQ.mRuby3).

[0214] Table 1. Capsid targeting peptides.

[0215]

[0216] To further analyze the selected variants, the AAV1 variant with the THTDDTR targeting peptide (SEQ ID NO:20) was packaged with the mTFP1 expression cassette (AAV1.THTD.mTFP1), the AAV2 variant with the SGGRYAE targeting peptide (SEQ ID NO:22) was packaged with the mNeonGreen expression cassette (AAV2.SGGR.NES_mNG), the AAV2 variant with the SKGQGTG targeting peptide (SEQ ID NO:21) was packaged with the mRuby3 expression cassette (AAV2.SKGQ.mRuby3), the AAV9 variant with the KMLSVVN targeting peptide (SEQ ID NO:24) was packaged with the Halo-tag expression cassette (AAV9KMLS.Halo-tag), and the AAV9 variant with the SLGSTKV targeting peptide (SEQ ID NO:23) was packaged with the mBFP2 expression cassette (AAV9SLGS.mBFP2).

[0217] The globus pallidus of two adult rhesus monkeys were injected with a total dose of 7.5E10 vg of certain capsid variants. Imaging studies were performed to determine the biodistribution and transport properties of each capsid after delivery to the globus pallidus. FISH imaging of 10 um z-stacks of tissue was used to localize mRNA from each fluorophore specific for its respective variant capsid ( Figure 1 , 4 , 7, 9, 11, 12, 15, 17, 18, 20, and 25). Epifluorescence imaging was used to localize proteins from each fluorophore specific for its respective variant capsid ( Figure 2 , 3 , 5, 6, 8, 10, 13, 14, 16, 17, 19, 21 to 24 and 26).

[0218] Example 2 - Injection of mouse with globus pallidus vector

[0219] Three mice received injections of different viruses in each hemisphere of the following designated anatomical structures: thalamus, intemal capsule / medial globuspallidus (IC / MGP), or deep cerebellar nuclei (DCN) as shown in Table 2. The different viruses were: RVC1023, pmAAV2.SGGR.NeonGreen; RVC1024, pmAAV2.SKGO.mRuby; and RVC1025, pmAAV1.THTD.mTFP. Mice were necropsied 22 days after injection and evaluated by epifluorescence microscopy ( Figures 27 to 37 ).

[0220] Table 2. Mouse injection paradigm for pallidum vectors.

[0221] Mouse ID ROA Left right vg / hemisphere 4279 thalamus NeonGreen mRuby 5.00E+09 4280 thalamus mRuby TFP 5.00E+09 4281 thalamus TFP NeonGreen 5.00E+09 4282 IC / MGP <![CDATA[NeonGree n ]]> <![CDATA[ m Ruby]]> 4.00E+09 4285 IC / MGP mRuby TFP 4.00E+09 4292 IC / MGP TFP NeonGreen 4.00E+09 4293 IC / MGP mRuby TFP 4.00E+09 A16 DCN <![CDATA[NeonGree n ]]> <![CDATA[ m Ruby]]> 4.00E+09 A17 DCN mRuby TFP 4.00E+09 A18 DCN TFP NeonGreen 4.00E+09

[0222] ***

[0223] According to the present disclosure, all methods disclosed and claimed herein can be carried out and implemented without excessive experimentation. Although the compositions and methods of the present invention have been described according to some preferred embodiments, it will be apparent to those skilled in the art that, without departing from the concept, spirit and scope of the present invention, changes can be applied to the steps or sequence of steps of the methods described herein and the methods described herein. More specifically, it will be apparent that certain medicaments related in both chemical and physiological aspects can be used to replace the medicaments described herein, while achieving the same or similar results. All such similar substitutions and changes apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

1. A modified adeno-associated virus (AAV) capsid protein, comprising a targeting peptide that targets a viral vector containing the modified AAV capsid protein to different organs or different brain structures, wherein the targeting peptide is three to ten amino acids in length.

2. The modified AAV capsid protein of claim 1, wherein the capsid protein has a sequence according to any one of SEQ ID NOs: 20, 19, and 21 to 24.

3. The modified AAV capsid protein of claim 1, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

4. The modified AAV capsid protein of claim 1, wherein the modified AAV capsid protein is derived from an AAV1 capsid protein (see SEQ ID NO: 1), wherein the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

5. The modified AAV capsid protein of claim 4, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptide are two or three amino acids long.

6. The modified AAV capsid protein of claim 5, wherein the linker sequence is SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

7. The modified AAV capsid protein of claim 6, wherein the modified AAV1 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:

4.

8. The modified AAV capsid protein of claim 1, wherein the modified AAV capsid protein is derived from an AAV2 capsid protein (see SEQ ID NO: 2), wherein the targeting peptide is inserted after residue 587 of the AAV2 capsid protein.

9. The modified AAV capsid protein of claim 8, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptide are two or three amino acids long.

10. The modified AAV capsid protein of claim 9, wherein the linker sequence is AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide.

11. The modified AAV capsid protein of claim 10, wherein the modified AAV2 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:

5.

12. The modified AAV capsid protein of claim 1, wherein the modified AAV capsid protein is derived from an AAV9 capsid protein (see SEQ ID NO: 3), wherein the targeting peptide is inserted after residue 588 of the AAV9 capsid protein.

13. The modified AAV capsid protein of claim 12, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptide are two or three amino acids long.

14. The modified AAV capsid protein of claim 13, wherein the linker sequence is AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

15. The modified AAV capsid protein of claim 14, wherein the modified AAV9 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:

6.

16. The modified AAV capsid protein of claim 1, wherein the targeting peptide comprises a sequence of up to ten amino acids in length having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10, 12, 14, 16, and 18.

17. The modified AAV capsid protein of claim 16, wherein the targeting peptide is seven amino acids in length.

18. The modified AAV capsid protein of any one of claims 1 to 17, wherein the different brain structures are globus pallidus, putamen, internal capsule, caudate nucleus, claustrum, substantia nigra, motor cortex, insula, temporal cortex, thalamus, hippocampus, subiculum, and deep cerebellar nuclei.

19. A nucleic acid comprising a sequence encoding the modified capsid protein of any one of claims 1 to 18.

20. A recombinant adeno-associated virus (rAAV) virus comprising the modified capsid protein of any one of claims 1 to 18.

21. A viral vector comprising a nucleic acid encoding the modified capsid protein of any one of claims 1 to 18.

22. The viral vector of claim 21, further comprising a nucleic acid sequence encoding a target nucleic acid.

23. The viral vector of claim 22, wherein the nucleic acid of interest is a therapeutic agent.

24. The viral vector of claim 23, wherein the therapeutic agent is a protein or an RNAi molecule.

25. A cell comprising the viral vector of any one of claims 21 to 24.

26. The cell of claim 25, wherein the cell is a mammalian cell.

27. The cell of claim 25, wherein the cell is a human cell.

28. The cell of claim 25, wherein the cell is in vitro.

29. The cell of claim 25, wherein the cell is in vivo.

30. A pharmaceutical composition comprising the viral vector of claim 20 and a pharmaceutically acceptable carrier.

31. A method of delivering an agent to distinct brain structures of a subject comprising administering to the subject the virus of claim 20.

32. The method of claim 31, wherein the agent is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein, or CRISPR system.

33. The method of any one of claims 31 to 32, wherein the administration is to the central nervous system.

34. The method of claim 33, wherein the administration is to the cisterna magna, the intraventricular space, the ependyma, the ventricle, the subarachnoid space, and / or the intrathecal space.

35. The method of claim 33, wherein the administration is to the globus pallidus.

36. The method of any one of claims 31 to 32, wherein the administration is systemic.

37. The method of any one of claims 31 to 36, wherein a plurality of viral particles are administered.

38. The method of claim 37, wherein the virus is present at about 1×10 6 About 1×10 18 The dose was administered at 1 vector genome / kilogram (vg / kg).

39. The method of claim 37, wherein the virus is administered at a dose of about 1×10 7 Up to 1×10 17 , about 1×10 8 Up to 1×10 16 , about 1×10 9 Up to 1×10 15 , about 1×10 10 Up to 1×10 14 , about 1×10 10 Up to 1×10 13 , about 1×10 10 Up to 1×10 13 , about 1×10 10 Up to 1×10 11 , about 1×10 11 Up to 1×10 12 , about 1×10 12 Up to ×10 13 , or about 1×10 13 Up to 1×10 14 vg / kg patient.

40. The method of any one of claims 31 to 39, wherein the subject is a human.

41. A method of treating a disease in a mammal comprising administering to said mammal the virus of claim 20.

42. The method of claim 41, wherein the disease is a neurodegenerative disease.

43. The method of claim 42, wherein the neurodegenerative disease is Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, Alzheimer's disease, polyglutamine repeat disease, or Parkinson's disease.

44. The method of claim 41, wherein the mammal is a human.

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