Adeno-associated virus vector variants

Abstract

The present invention provides a vector containing a sequence encoding a targeting peptide that delivers a preparation to a specific substructure in the brain, and a targeting peptide.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 939,315 filed November 22, 2019, and U.S. Provisional Application No. 63 / 084,709 filed September 29, 2020, the full contents of both of these applications are incorporated herein by reference in their entirety.

[0003] Reference to the sequence list

[0004]

[0002] The present application includes a list of sequences submitted via EFS-Web in ASCII format, the full text of which is incorporated by reference. The filename of the said ASCII copy created on November 19, 2020, is CHOPP0038WO_ST25.txt and is 63.8 kilobytes in size. Background Technology

[0005] 1. Technology Field

[0006]

[0003] The present invention generally relates to the fields of medicine, virology, and neurology. More particularly, it relates to a targeting peptide that targets the delivery of a viral vector to a unique structure in the brain.

[0007] 2. Entry in related fields

[0008]

[0004] Different strategies have been developed to generate AAV vector variants involving rational design and directed advancement. The rational design approach leverages knowledge of the AAV capsid to create targeted modifications to the capsid to alter transduction efficiency or specificity, such as tyrosine mutations on the capsid surface to increase transduction efficiency. The directed advancement approach requires no prior knowledge of the capsid structure and is performed through random mutagenesis, capsid shuffling, or random peptide insertion. These strategies generally utilize in vitro systems or mice ideal for cell-based or mouse studies, but do not imply clinical application. In fact, no AAV variant specifically or efficiently targets unique brain structures. As such, there is a need for AAV variants capable of targeting unique primate brain structures.

[0009] Summary of the Invention

[0010]

[0005] In the present invention, a viral vector comprising a modified capsid is provided, wherein the modified capsid comprises at least one amino acid sequence that targets the viral vector to a unique brain structure.

[0011]

[0006] In one embodiment, a modified adeno-associated virus (AAV) capsid protein is provided, comprising a targeting peptide that targets a viral vector containing the modified AAV capsid protein to a specific organ or brain structure, said targeting peptide being 3 to 10 amino acids in length. 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.

[0012]

[0007] In some aspects, the modified AAV capsid protein is derived from the AAV1 capsid protein (see SEQ ID NO. 138), and the targeting peptide is inserted after residue 590 of the AAV1 capsid protein. In some aspects, the targeting peptide is flanked by a linker sequence, and the linker sequence on each side of the targeting peptide is 2 or 3 amino acids long. In some aspects, the linker sequence is an SSA on the N-terminal side of the targeting peptide and an AS on the C-terminal side of the targeting peptide. In some aspects, the modified AAV1 capsid protein has a sequence that is at least 95% identical to SEQ ID NO. 141.

[0013]

[0008] In some aspects, the modified AAV capsid protein is derived from the AAV2 capsid protein (see SEQ ID NO. 139), and the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. In some aspects, the targeting peptide is flanked by a linker sequence, and the linker sequence on each side of the targeting peptide is 2 or 3 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 that is at least 95% identical to SEQ ID NO. 142.

[0014]

[0009] In some aspects, the modified AAV capsid protein is derived from the AAV9 capsid protein (see SEQ ID NO. 140), and the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. In some aspects, the targeting peptide is flanked by a linker sequence, and the linker sequence on each side of the targeting peptide is 2 or 3 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 that is at least 95% identical to SEQ ID NO. 143.

[0015]

[0010] In some aspects, the targeting peptide comprises a sequence of up to 10 amino acids in length having an amino acid sequence selected from the group consisting of SEQ ID NOs 1-137 and 144. In some aspects, the targeting peptide is 7 amino acids in length.

[0016]

[0011] In some aspects, the unique brain structures are the brainstem, caudate, cerebellar cortex, cerebral cortex, ependymal space, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. In certain aspects, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is selected from those listed in Table 1 to target the corresponding brain structures. In certain aspects, the modified AAV capsid protein is the modified AAV2 capsid protein, and the targeting peptide is selected from those listed in Table 2 to target the corresponding brain structures. In certain aspects, the modified AAV capsid protein is the modified AAV9 capsid protein, and the targeting peptide is selected from those listed in Table 3 to target the corresponding brain structures.

[0017]

[0012] In some aspects, the specific organ is the brain, kidney, heart, liver, gonads, spleen, or liver. In certain aspects, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is selected from those listed in Table 4 to target the corresponding organ. In certain aspects, the modified AAV capsid protein is the modified AAV2 capsid protein, and the targeting peptide is selected from those listed in Table 5 to target the corresponding organ. In certain aspects, the modified AAV capsid protein is the modified AAV9 capsid protein, and the targeting peptide is selected from those listed in Table 6 to target the corresponding organ.

[0018]

[0013] In one embodiment, the present invention provides a nucleic acid comprising a sequence encoding a modified capsid protein of any one of the embodiments of the present invention.

[0019]

[0014] In one embodiment, the present invention provides a recombinant adeno-associated virus (rAAV) comprising a modified capsid protein of any one of the embodiments of the present invention. In some aspects, combinations of rAAVs are provided. For example, combinations of rAAV having a modified AAV1 capsid protein and a targeting peptide of SEQ ID NO. 21, rAAV having a modified AAV2 capsid protein and a targeting peptide of SEQ ID NO. 53, rAAV having a modified AAV2 capsid protein and a targeting peptide of SEQ ID NO. 80, and rAAV having a modified AAV9 capsid protein and a targeting peptide of SEQ ID NO. 113 are provided.

[0020]

[0015] In one embodiment, the present invention provides a viral vector comprising a sequence 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 nucleic acid of interest. In some aspects, the nucleic acid of interest is a therapeutic agent. In some aspects, the therapeutic agent is an enzyme or an RNAi molecule.

[0021]

[0016] In one embodiment, the present invention provides a cell comprising any one of the virus vectors of the embodiments of the present invention. In some aspects, the cell is a mammalian cell, e.g., a human cell. In some aspects, the cell is an in vitro or in vivo cell.

[0022]

[0017] In one embodiment, the present invention provides a pharmaceutical composition comprising a virus vector of an embodiment of the present invention and a pharmaceutically acceptable carrier.

[0023]

[0018] In one embodiment, the present invention provides a method for delivering a formulation to a specific brain structure of a subject, said method comprising the step of administering the virus of an embodiment of the present invention to said subject. In some aspects, the specific brain structure is the brainstem, caudate, cerebellar cortex, cerebral cortex, ependymal space, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. In certain aspects, rAAV having a modified AAV1 capsid protein is used, and a targeting peptide is selected from those listed in Table 1 to target the corresponding brain structure. In certain aspects, rAAV having a modified AAV2 capsid protein is used, and a targeting peptide is selected from those listed in Table 2 to target the corresponding brain structure. In certain aspects, rAAV having a modified AAV9 capsid protein is used, and a targeting peptide is selected from those listed in Table 3 to target the corresponding brain structure. In various aspects, any combination of rAAVs is used. For example, a combination of rAAV having a modified AAV1 capsid protein and a targeting peptide of SEQ ID NO. 21, rAAV having a modified AAV2 capsid protein and a targeting peptide of SEQ ID NO. 53, rAAV having a modified AAV2 capsid protein and a targeting peptide of SEQ ID NO. 80, and rAAV having a modified AAV9 capsid protein and a targeting peptide of SEQ ID NO. 113 is used.

[0024]

[0019] In one embodiment, the present invention provides a method for delivering a preparation to a specific organ of a subject, said method comprising the step of administering the virus of an embodiment of the present invention to said subject. In some aspects, the organ is the brain, kidney, heart, liver, gonad, spleen, or liver. In certain aspects, rAAV having a modified AAV1 capsid protein is used, and a targeting peptide is selected from those listed in Table 4 to target the corresponding organ. In certain aspects, rAAV having a modified AAV2 capsid protein is used, and a targeting peptide is selected from those listed in Table 5 to target the corresponding organ. In certain aspects, rAAV having a modified AAV9 capsid protein is used, and a targeting peptide is selected from those listed in Table 6 to target the corresponding organ. In various aspects, any combination of rAAV is used.

[0025]

[0020] In some aspects, the agent is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein, or CRISPR system. In some aspects, administration is to the central nervous system. In some aspects, administration is to the logarithmic cistern, intraventricular space, ependymal space, ventricles, subarachnoid space, and / or intravertebral space. In some aspects, the ventricles are the main lateral ventricle, and / or caudate lateral ventricle, and / or right ventricle, and / or left ventricle, and / or right main lateral ventricle, and / or left main lateral ventricle, and / or right caudate lateral ventricle, and / or left caudate lateral ventricle.

[0026]

[0021] In some aspects, a large number of virus particles are administered. In some aspects, the virus is approximately 1 × 10⁶ per kilogram. 6 to about 1×10 18 It is administered at a dose of the vector genome (vg / kg). In some modalities, the virus is approximately 1 x 10⁶ 7 -1x10 17 , approximately 1x108 -1x10 16 , approximately 1x10 9 -1x10 15 , approximately 1x10 10 -1x10 14 , approximately 1x10 10 -1x10 13 , approximately 1x10 10 -1x10 13 , approximately 1x10 10 -1x10 11 , approximately 1x10 11 -1x10 12 , approximately 1x10 12 -x10 13 , or about 1x10 13 -1X10 14 It is administered at a dose of vg / kg of patient. In some aspects, the subject is a human.

[0027]

[0022] In one embodiment, the present invention provides a method for treating a disease in a mammal, comprising the step of administering the virus of an embodiment of the present invention to the mammal. In some embodiments, the disease is a neurodegenerative disease. In some aspects, the neurodegenerative disease is Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, Alzheimer's disease, polyglutamine recurrent disease, or Parkinson's disease. In some aspects, the mammal is a human.

[0028]

[0023] With respect to specific components as used herein, “essentially absent” is used to mean that none of the specific components are intentionally formulated into the composition or are present only as contaminants or in trace amounts. Accordingly, the total amount of the specific component resulting from any unintended contamination of the composition is less than 0.05%, preferably 0.01%. A composition in which the amount of the specific component cannot be detected by standard analytical methods is most preferred.

[0029]

[0024] In the present specification, the singular article (“a” or “an”) may mean one or more. In the claims of the present invention, the singular article (“a” or “an”) may mean one or more when used with the word “comprising”.

[0030]

[0025] The use of the term “or” in a claim is used to mean “and / or” unless it explicitly refers only to an alternative, or even if the specification supports a definition referring only to an alternative and “and / or,” unless the alternatives are mutually exclusive. In this document, the term “another” may mean at least a second or more.

[0031]

[0026] Throughout this application, the term “approximately” is used to indicate that a value includes variations in inherent error of the device, the method used to measure the value, or variations existing between the subjects of study, and that the value is within 10% of the said value.

[0032]

[0027] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments presenting preferred embodiments of the present invention are provided merely as examples, as it is obvious to those skilled in the art that various changes and modifications can be made within the concept and scope of the present invention from the detailed description. Brief explanation of the drawing

[0033]

[0028] The following drawings form part of this specification and are incorporated to further demonstrate specific aspects of the invention. The invention may be better understood by reference to one or more of these drawings, together with the detailed description of specific embodiments provided herein.

[0029] Fig. 1. Schematic of AAV peptide display library.

[0030] Fig. 2Schematic of in vivo screening strategy.

[0031] Fig. 3. Graph of input library diversity provided. Diversity of the input virus library measured from aliquots of AAV1, AAV2, and AAV9 viral vectors prior to Round 1 ICV injection.

[0032] Fig. 4. Graph of barcode enrichment by round provided. Total number of unique barcodes recovered after Round 1 and Round 2 intraperitoneal analysis from Rhus maculatus for each collected tissue. Represents Round 2 values ​​for DNA and RNA.

[0033] Fig. 5. Illustration of round-by-round enrichment of barcodes in AAV1, AAV2, and AAV9 serotypes for the cerebellar cortex.

[0034] Fig. 6. Illustration of AAV9 1999 enrichment. To depict the heatmap of AAV9 barcode enrichment, cells are colored according to the percentage of barcodes detected from a specified tissue. Barcodes recovered from DNA are shown on the upper left, and barcodes recovered from RNA are shown on the upper right.

[0035] Fig. 7a-c. Heatmap depiction of opool barcode enrichment from AAV1 (Fig. 7a), AAV2 (Fig. 7b), and AAV9 (Fig. 7c).

[0036] Fig. 8. Confirmation of AAV9 1999 in vivo in Rhesus macaques. An eGFP-expressing construct was packaged into AAV9 1999 driven by a CAG promoter. 1.5E13 vg of AAV9 1999 was delivered to 5-year-old female Rhesus macaques by ICV injection. Representative images of the H&E-stained cerebellum depicting the transduction pattern of AAV9 1999 are shown.

[0037] Fig. 9a-d.In vivo mouse confirmation of AAV9 1999. An eGFP-expressing construct was packaged into AAV9 1999 driven by a CAG promoter. AAV9 1999 and AAV9 capsid containing the eGFP construct were delivered to C57BL / 6 p0 mouse pups by ICV injection at 1E10 vg. Representative images of the eGFP fluorescence signal are the forebrain (Fig. 9a), forebrain thalamus section (Fig. 9b), S1 cortical section (Fig. 9c, left), hippocampal section (Fig. 9c, middle), cerebellar thalamus section (Fig. 9c, right), and lumbar spinal cord coronal section (Fig. 9d).

[0038] Fig. 10a-c. Fluorescence images of the lateral ventricle (a), fourth ventricle (b), and meninges (c) in vivo with an AAV mixture of rhesus macaques.

[0039] Fig. 11a-c. Fluorescence images of cochlear rotation (a), internal hair cells (b), cortical organelles (c) and distal modulus (c) after cochlear administration of AAV9 1999 capsid containing eGFP construct to mice. Specific details for implementing the invention

[0034] Detailed description of the invention

[0035]

[0040] The present invention provides a viral vector comprising a modified capsid, wherein the modified capsid comprises at least one amino acid sequence that targets the viral vector to a specific brain structure. In certain embodiments, the brain structure is the brainstem, caudate, cerebellar cortex, cerebral cortex, ependymal, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. Targeting peptides for each brain structure are provided in Tables 1 through 3.

[0036]

[0041] In certain embodiments, the viral vector is an adeno-associated virus vector (AAV). In certain embodiments, the AAV is AAV1, AAV2, or AAV9. An exemplary wild-type reference AAV1 capsid protein sequence is provided at SEQ ID NO. 138. An exemplary wild-type reference AAV2 capsid protein sequence is provided at SEQ ID NO. 139. An exemplary wild-type reference AAV9 capsid protein sequence is provided at SEQ ID NO. 140. In certain aspects, a 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 at SEQ ID NO. 141, which shows the insertion of a targeting peptide after position 590 as SSAX7AS, where the leading SSA and trailing AS are linker sequences and X7 represents the targeting peptide. An exemplary modified AAV2 capsid protein sequence is provided at SEQ ID NO. 142, which shows a targeting peptide insertion after position 587 as AAAX7AA, where the leading AAA and trailing AA are linker sequences and X7 represents the targeting peptide. An exemplary modified AAV9 capsid protein sequence is provided at SEQ ID NO. 143, which shows a targeting peptide insertion after position 588 as AAAX7AS, where the leading AAA and trailing AS are linker sequences and X7 represents the targeting peptide.

[0037] [Table 1]

[0038] AAV1 targeting peptides for each brain structure.

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] [Table 2]

[0048] AAV2-targeting peptides for each brain structure.

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] [Table 3]

[0059] AAV9-targeting peptides for each brain structure.

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] [Table 4]

[0069] AAV1-targeting peptides for various organs.

[0070]

[0071] [Table 5]

[0072] AAV2-targeting peptides for various organs.

[0073]

[0074]

[0075] [Table 6]

[0076] AAV9-targeting peptides for various organs.

[0077]

[0078] I. Adeno-associated virus (AAV) vectors

[0079]

[0042] Adeno-associated viruses (AAVs) are small, non-pathogenic viruses of the parvoviridae family. To date, numerous serologically distinct AAVs have been identified, and more than 12 AAVs have been isolated from humans or primates. AAVs are distinguished from other members of the family by their reliance on helper viruses for replication.

[0080]

[0043] The AAV genome can exist extrachromosomally without being integrated into the host cell genome; it possesses a broad host range; it transduces both dividing and non-dividing cells in vitro and in vivo and maintains high levels of expression of the transduced genes. AAV viral particles are heat-stable; resistant to changes in solvents, detergents, pH, and temperature; and can be column-purified and / or concentrated by a CsCl gradient or other means. The AAV genome contains positive or negatively detected single-stranded deoxyribonucleic acid (ssDNA). The approximately 4.7 kb genome of AAV consists of a single fragment of single-stranded DNA with positive or negative polarity. The ends of the genome are short inversion end repeats (ITRs) that can fold into a hairpin structure and act as the origin of viral DNA replication.

[0081]

[0044] The AAV “genome” refers to the recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form an AAV particle. An AAV particle typically contains an AAV genome packaged with an AAV capsid protein. When a recombinant plasmid is used to construct or manufacture a recombinant vector, the AAV vector genome does not contain a “plasmid” portion that does not correspond to the vector genome sequence of the recombinant plasmid. The non-vector genome portion of the recombinant plasmid is referred to as the “plasmid backbone,” which is important for plasmid cloning and amplification; while it is a necessary process for plasmid amplification and production, it is not itself packaged or encapsulated into a viral particle. Therefore, the AAV vector “genome” refers to the nucleic acid packaged or encapsulated by the AAV capsid protein.

[0082]

[0045] AAV virions (particles) are non-enveloped, icosahedral particles with a diameter of approximately 25 nm containing an AAV capsid. AAV particles possess icosahedral symmetry composed of three related capsid proteins—VP1, VP2, and VP3—that interact together to form the capsid. The genomes of most native AAVs contain two open read frames (ORFs), often referred to as the left ORF and the right ORF. The right ORF commonly encodes the capsid proteins VP1, VP2, and VP3. These proteins are typically found in a ratio of 1:1:10, respectively, but can exist in varying proportions and all originate from the right ORF. The VP1, VP2, and VP3 capsid proteins differ from one another in that they utilize alternative splicing and specific start codons. Deletion analyses have shown that the removal or alteration of VP1, which is decoded from the generally spliced ​​message, reduces the yield of infectious particles. Mutations within the VP3 coding region prevent the generation of any single-stranded progeny DNA or infectious particles. In certain embodiments, the genome of the AAV particle encodes one, two, or all three VP1, VP2, and VP3 polypeptides.

[0083]

[0046] The left ORF commonly encodes the non-structural Rep proteins, Rep 40, Rep 52, Rep 68, and Rep 78, which are involved in replication and transcription regulation in addition to the generation of single-stranded progeny genomes. Two of the Rep proteins are associated with the preferential integration of the AAV genome into the q-arm region of human chromosome 19. Rep68 / 78 have been shown to possess DNA and RNA helicase activity as well as NTP binding activity. Some Rep proteins possess multiple potential phosphorylation sites as well as nuclear localization signals. In certain embodiments, the genome of the AAV (e.g., rAAV) encodes some or all of the Rep proteins. In certain embodiments, the genome of the AAV (e.g., rAAV) is not Rep proteinized. In certain embodiments, one or more of the Rep proteins may be trans-transmitted and are therefore not included in an AAV particle containing nucleic acids encoding polypeptides.

[0084]

[0047] The ends of the AAV genome contain short inverted end repeats (ITRs) that have the potential to fold into a T-shaped hairpin structure, acting as the origin for viral DNA replication. Thus, the AAV genome contains one or more (e.g., a pair) ITR sequences that flank the single-stranded viral DNA genome. ITR sequences are typically about 145 bases long each. Within the ITR region, two elements have been described, and they are thought to play a central role in the function of the ITR, the GAGC repeat motif, and the end-disconnection site (trs). The repeat motif was shown to bind to Rep when the ITR is in a linear or hairpin form. This binding is thought to be a binding to the Rep68 / 78 site for cleavage in the trs, occurring in a site- and strand-specific manner. In addition to their roles in replication, these two elements appear to be important for viral integration. The integration locus on chromosome 19 contains a Rep binding site with adjacent trs. These elements were found to function and be necessary for locus-specific integration.

[0085]

[0048] As a modifier for recombinant viruses, such as lenti- or parvovirus (e.g., AAV) vectors, the term “recombinant” generally implies that the composition has been manipulated (i.e., processed) in a manner not found in nature. Specific examples of recombinant vectors, such as AAV, retrovirus, or lentivirus vectors, are cases where nucleic acid sequences not normally present in the wild-type viral genome are inserted into the viral genome. Examples of recombinant nucleic acid sequences are cases where a nucleic acid (e.g., a gene) encodes a repressor RNA cloned into a vector that has or lacks intron regions where the gene normally binds within the viral genome, such as 5', 3', and / or genes. While the term “recombinant” is not always used in relation to vectors such as viral vectors and sequences such as polynucleotides, “recombinant” forms including nucleic acid sequences, polynucleotides, transposable genes, etc., are explicitly included in any of the above omissions.

[0086]

[0049] A recombinant viral "vector" is derived from the wild-type genome of a virus by using molecular methods to remove portions of the wild-type genome from the virus and replacing them with non-native nucleic acids, such as nucleic acid sequences. Typically, for example in the case of AAV, one or both inversion terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. "Recombinant" viral vectors (e.g., rAAV) are distinguished from the viral genome (e.g., AAV) genome because portions of the viral genome are replaced with non-native sequences relative to the viral genome nucleic acids, such as nucleic acids encoding transactivators, nucleic acids encoding repressive RNA, or nucleic acids encoding therapeutic proteins. Therefore, the incorporation of these non-native nucleic acid sequences defines the viral vector as a "recombinant" vector, and in the case of AAV, it may be referred to as an "rAAV vector."

[0087]

[0050] In a specific embodiment, the AAV (e.g., rAAV) comprises two ITRs. In a specific embodiment, the AAV (e.g., rAAV) comprises a pair of ITRs. In a specific embodiment, the AAV (e.g., rAAV) comprises a pair of ITRs that flank (i.e., at the respective 5' and 3' ends) a nucleic acid sequence encoding at least a polypeptide having function or activity.

[0088]

[0051] AAV vectors (e.g., rAAV vectors) may be packaged and are referred to herein as "AAV particles" for subsequent infection (transduction) of cells in vitro, in vitro, or in vivo. When a recombinant AAV vector is encapsulated or packaged into AAV particles, the particles may also be referred to as "rAAV particles." In certain embodiments, the AAV particles are rAAV particles. rAAV particles often contain an rAAV vector or a portion thereof. rAAV particles may be one or more rAAV particles (e.g., multiple AAV particles). rAAV particles typically contain a protein that encapsulates or packages the rAAV vector genome (e.g., capsid protein). It should be noted that a reference to an rAAV vector may also be used to refer to rAAV particles.

[0089]

[0052] Any suitable AAV particle (e.g., rAAV particle) may be used in the method or use herein. The rAAV particle and / or genome included herein may be derived from any suitable serotype or strain of AAV. The rAAV particle and / or genome included herein may be derived from two or more serotypes or strains of AAV. Thus, rAAV may contain proteins and / or nucleic acids of any serotype or strain of AAV, or parts thereof, said AAV particle is suitable for infection and / or transduction of mammalian cells. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, and AAV-2i8.

[0090]

[0053] In a specific embodiment, a plurality of rAAV particles comprises particles of the same strain or serotype (or subgroup or variant) or particles derived therefrom. In a specific embodiment, a plurality of rAAV particles comprises a mixture of two or more different rAAV particles (e.g., of different serotypes and / or strains).

[0091]

[0054] The term “serotype” as used herein is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological characteristics are determined based on the absence of cross-reactivity between antibodies to one AAV compared to other AAVs. Such cross-reactivity differences are generally due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of the AAV serotype). Despite the possibility that an AAV variant containing a capsid variant may not be serologically distinct from a reference AAV or other AAV serotypes, it differs in at least one nucleotide or amino acid residue compared to the reference or other AAV serotypes.

[0092]

[0055] In a specific embodiment, an rAAV vector based on a first serotype genome corresponds to a serotype of one or more capsid proteins packaging the vector. For example, a serotype of one or more AAV nucleic acids (e.g., ITR) containing an AAV vector genome corresponds to a serotype of a capsid containing an rAAV particle.

[0093]

[0056] In a specific embodiment, the rAAV vector genome may be based on an AAV (e.g., AAV2) serotype genome that is distinct from the serotype of one or more AAV capsid proteins packaging the vector. For example, the rAAV vector genome may contain nucleic acids derived from AAV2 (e.g., ITR), while at least one of the three capsid proteins is 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 a variant thereof.

[0094]

[0057] In a specific embodiment, the rAAV particle or its vector genome associated with the reference serotype is at least 60% (polynucleotide, polypeptide, or subsequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 particle) for example , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) have polynucleotides, polypeptides, or subsequences comprising or composed of identical sequences. In certain embodiments, the rAAV particle or its vector genome associated with a reference serotype has at least 60% (capsid or ITR sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotypes) for example , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) have a capsid or ITR sequence containing the same sequence or composed of these.

[0095]

[0058] In a specific embodiment, the method of the present invention comprises the use, administration, or delivery of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74, or rAAV-2i8 particles.

[0096]

[0059] In certain embodiments, the method of the present invention comprises the use, administration, or delivery of rAAV2 particles. In certain embodiments, the rAAV2 particles comprise an AAV2 capsid. In certain embodiments, the rAAV2 particles comprise one or more capsid proteins (e.g., VP1, VP2, and / or Vp3) that are at least 60%, 65%, 70%, 75% or more identical to the corresponding capsid protein of an indigenous 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 to the corresponding capsid protein of an indigenous or wild-type AAV2 particle. In a specific embodiment, the rAAV2 particle comprises one VP1, VP2, and / or VP3 capsid protein that is at least 75% identical to the corresponding capsid protein of the 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. In a specific embodiment, the rAAV2 particle is a variant of the native or wild-type AAV2 particle. In some aspects, one or more capsid proteins of an AAV2 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to the capsid protein(s) of the native or wild-type AAV2 particle.

[0097]

[0060] In a specific embodiment, the rAAV9 particle comprises an AAV9 capsid. In a specific embodiment, the rAAV9 particle comprises one or more capsid proteins (e.g., VP1, VP2 and / or Vp3) that are at least 60%, 65%, 70%, 75% or more identical to the corresponding capsid protein of the native or wild-type AAV9 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. In a specific embodiment, the rAAV9 particle comprises one VP1, VP2, and / or VP3 capsid protein that is at least 75% identical to the corresponding capsid protein of the native or wild-type AAV9 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. In a specific embodiment, the rAAV9 particle is a variant of the native or wild-type AAV9 particle. In some aspects, one or more capsid proteins of an AAV9 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to the capsid protein(s) of the native or wild-type AAV9 particle.

[0098]

[0061] In a specific embodiment, the rAAV particle is at least 75% identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., to 100% identical to one or two ITRs (e.g., a pair of ITRs) They include, provided that they possess one or more desired ITR functions (e.g., DNA replication; integration of AAV DNA into the host cell genome; and / or, in some cases, the ability to form a hairpin that enables packaging).

[0099]

[0062] In a specific embodiment, the rAAV2 particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% identical to the corresponding ITR of the 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, provided that they possess one or more desired ITR functions (e.g., DNA replication; integration of AAV DNA into the host cell genome; and / or, in some cases, the ability to form a hairpin that enables packaging).

[0100]

[0063] In a specific embodiment, the rAAV9 particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% identical to the corresponding ITR of the 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, provided that they possess one or more desired ITR functions (e.g., DNA replication; integration of AAV DNA into the host cell genome; and / or, in some cases, the ability to form a hairpin that enables packaging).

[0101]

[0064] The rAAV particle may comprise an ITR having any suitable number of “GAGC” repeats. In a specific embodiment, the ITR of the AAV2 particle comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more “GAGC” repeats. In a specific embodiment, the rAAV2 particle comprises an ITR comprising 3 “GAGC” repeats. In a specific embodiment, the rAAV2 particle comprises an ITR having fewer than 4 “GAGC” repeats. In a specific embodiment, the rAAV2 particle comprises an ITR having more than 4 “GAGC” repeats. In a specific embodiment, the ITR of the rAAV2 particle comprises a Rep binding site, wherein the 4th nucleotide within the first two “GAGC” repeats is C rather than T.

[0102]

[0065] DNA of an exemplary suitable length may be incorporated into an rAAV vector for packaging / encapsulation into rAAV particles that may be about 5 kilobases (kb) or less. In certain embodiments, the length of the DNA is less than about 5 kb, less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, or less than about 2.5 kb.

[0103]

[0066] rAAV vectors containing nucleic acid sequences directing the expression of RNAi or polypeptides can be generated using suitable recombinant techniques known in the art (e.g., see Sambrook et al. (e.g., 1989). Recombinant AAV vectors are typically packaged into transfection-qualified AAV particles or replicated using an AAV virus packaging system. Transfection-qualified AAV particles can bind to mammalian cells, enter them, and subsequently deliver nucleic acid cargo (heterogeneous genes) into the cell nucleus. Thus, intact transfection-qualified rAAV particles are configured to transfect mammalian cells. rAAV particles configured to transfect mammalian cells are often not replication-qualified and require additional protein machinery for self-replication. Therefore, rAAV particles configured to transfect mammalian cells are processed to bind to mammalian cells, enter them, and deliver nucleic acids to the cells, where the nucleic acids for delivery are often located between a pair of AAV ITRs in the rAAV genome.

[0104]

[0067] Suitable host cells for generating transduction-eligible AAV particles include, but are not limited to, microbial, yeast, insect, and mammalian cells that may be or have been used as recipients of a heterologous rAAV vector. Cells from a stable human cell line, HEK293 (e.g., readily available through the depositary (the American Type Culture Collection under Accession Number ATCC CRL1573)) may be used. In a specific embodiment, recombinant AAV particles are generated using a modified human embryonic kidney cell line (e.g., HEK293) that is transformed with an adenovirus type 5 DNA fragment and expresses adenovirus E1a and E1b genes. The modified HEK293 cell line is easily transfected and provides a simple platform, particularly for generating rAAV particles. Methods for generating high-titer AAV particles capable of transducing mammalian cells are known in the art. For example, AAV particles can be manufactured as described in the literature (see Wright, 2008 and Wright, 2009).

[0105]

[0068] In certain embodiments, AAV helper function is introduced into host cells by transfecting host cells with an AAV helper construct prior to or simultaneously with transfection with an AAV expression vector. Thus, AAV helper constructs are sometimes used to provide at least transient expression of the AAV rep and / or cap genes to compensate for lost AAV function required for productive AAV transduction. AAV helper constructs often lack an AAV ITR and cannot replicate or package themselves. These constructs may be in the form of plasmids, phages, transposons, cosmids, viruses, or virions. A number of commonly used AAV helper constructs, such as the plasmids pAAV / Ad and pIM29+45, which encode both Rep and Cap expression products, have been described. A number of other vectors encoding Rep and / or Cap expression products are known.

[0106]

[0069] An “expression vector” is a specialized vector containing a gene or nucleic acid sequence along with the necessary regulatory regions required for expression in a host cell. An expression vector may contain at least a replication origin for reproduction in a cell and may optionally contain additional elements, e.g., a heterogeneous nucleic acid sequence, expression control elements (e.g., promoter, enhancer), introns, ITR(s), and polyadenylation signals.

[0107] II. Therapeutic Agents

[0108]

[0070] In some embodiments, nucleic acids may be introduced into mammalian cells or target tissues using a viral gene delivery method. The method may be used to administer nucleic acids encoding repressive RNA, non-coding RNA, and / or therapeutic proteins into cells within a culture or host organism.

[0109] A. Inhibitory RNA

[0110]

[0071] “RNA interference (RNAi)” is a sequence-specific, post-transcriptional gene silencing process initiated by siRNA. During RNAi, siRNA induces the degradation of target mRNA and, consequently, sequence-specifically inhibits gene expression.

[0111]

[0072] “Repressive RNA,” “RNAi,” “small interfering RNA,” “short interfering RNA,” or “siRNA” molecules, “short hairpin RNA,” or “shRNA” molecules, or “miRNA” are RNA duplexes of nucleotides targeted to the nucleic acid sequence of interest. The term “siRNA,” as used herein, is a general term encompassing a subset of shRNA and miRNA. “RNA duplex” refers to a structure formed by complementary pairing between two regions of an RNA molecule. siRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the targeted gene. In certain embodiments, the siRNA is targeted to a sequence encoding huntingtin. In some embodiments, the length of the siRNA duplex is less than 30 base pairs. In some embodiments, the duplex may be about 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 base pairs long. In some embodiments, the length of the duplex is 19 to 25 base pairs long. In certain embodiments, the length of the duplex is 19 or 21 base pairs long. The RNA duplex portion of the siRNA may be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion located between the two sequences forming the duplex. The length of the loop may vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide lengths. In certain embodiments, the loop is 18 nucleotide lengths. The hairpin structure may also contain 3′ and / or 5′ overhang portions. In some embodiments, the overhang is 3′ and / or 5′ overhang and is 0, 1, 2, 3, 4, or 5 nucleotide lengths.

[0112]

[0073] shRNA consists of a stem-loop structure designed to contain a 5' flanking region, an siRNA region fragment, a loop region, a 3' siRNA region, and a 3' flanking region. Most RNAi expression strategies have utilized short-hairpin RNA (shRNA) driven by strong poIII-based promoters. While many shRNAs have demonstrated effective knockdown of target sequences in vitro and in vivo, some shRNAs that demonstrated effective knockdown of target genes were also found to be toxic in vivo.

[0113]

[0074] miRNA is a small cellular RNA (~22 nt) processed from a precursor stem loop transcript. Known miRNA stem loops can be modified to contain RNAi sequences specific to the gene of interest. Since miRNA molecules are expressed endogenously, miRNA molecules may be preferred over shRNA molecules. Thus, miRNA molecules are unlikely to induce the dsRNA-responsive interferon pathway, and they are processed more efficiently than shRNA and have been shown to silence 80% more effectively.

[0114]

[0075] A recently discovered alternative approach is to use artificial miRNAs (pri-miRNA scaffolds that transfer siRNA sequences) as RNAi vectors. Artificial miRNAs are more naturally similar to endogenous RNAi substrates and can be more compliant with Pol-II transcription (e.g., enabling tissue-specific expression of RNAi) and polycistron strategies (e.g., enabling the transfer of multiple siRNA sequences). See U.S. Patent No. 10,093,927 cited by reference.

[0115]

[0076] The transcription unit of “shRNA” consists of sense and antisense sequences connected by loops of unpaired nucleotides. shRNA is released from the nucleus by Exportin-5 and, once it enters the cytoplasm, is processed by Dicer to produce functional siRNA. The “miRNA” stem loop consists of sense and antisense sequences connected by loops of unpaired nucleotides, which are typically expressed as part of a larger primary transcript (pri-miRNA), said primary transcript is cleaved by the Drosha-DGCR8 complex to produce an intermediate known as pre-miRNA, which is then released from the nucleus by Exportin-5 and, once it enters the cytoplasm, is processed by Dicer to produce functional siRNA. As used herein interchangeably, “artificial miRNA” or “artificial miRNA shuttle vector” refers to a primary miRNA transcript having a region of a duplex stem loop (at least about 9–20 nucleotides) that is cleaved by Drosha and Dicer processing, and is replaced by an siRNA sequence for a target gene while maintaining structural elements within the stem loop necessary for effective Drosha processing. The term “artificial” is derived from the fact that a flanking sequence (~35 nucleotides upstream and ~40 nucleotides downstream) occurs at the restriction enzyme site within the siRNA’s multiple cloning site. As used herein, the term “miRNA” encompasses both naturally occurring miRNA sequences and artificially generated miRNA shuttle vectors.

[0116]

[0077] siRNA may be encoded by a nucleic acid sequence, said nucleic acid sequence may also include a promoter. The nucleic acid sequence may also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a 6 T sequence.

[0117]

[0078] In designing RNAi, there are several factors that need to be considered, such as the properties of the siRNA, the duration of the silencing effect, and the selection of the delivery system. The siRNA introduced into an organism to produce an RNAi effect typically contains an exon sequence. Additionally, since the RNAi process is homology-dependent, the sequence must be carefully selected to maximize gene specificity while minimizing the potential for cross-interference between homologous but gene-specific sequences. Preferably, the siRNA exhibits 80%, 85%, 90%, 95%, over 98%, or even 100% identity between the siRNA sequence and the gene to be repressed. Sequences identical to the target gene by less than approximately 80% are substantially less effective. Therefore, the greater the homology between the siRNA and the gene to be repressed, the less the expression of unrelated genes will be affected.

[0118]

[0079] In addition, the size of the siRNA is an important consideration. In some embodiments, the present invention relates to an siRNA molecule capable of regulating gene expression, comprising at least about 19 to 25 nucleotides. In the context of the present invention, the siRNA is preferably 500, 200, 100, 50, or 25 nucleotide lengths. More preferably, the siRNA is about 19 to about 25 nucleotide lengths.

[0119]

[0080] siRNA targets generally refer to polynucleotides containing a region encoding a polypeptide, or a polynucleotide region regulating replication, transcription, or translation or other processes important for polypeptide expression, or a polynucleotide containing both a region encoding a polypeptide and a region operatively linked to regulate expression. Any gene expressed within a cell can be targeted. Preferably, the target gene is involved in or related to the progression of cellular activity of a specific target of interest, which is important for the disease or a subject of study.

[0120] B. Non-coding RNA

[0121]

[0081] As demonstrated by cDNA cloning projects and genome tiling arrays, more than 90% of the human genome undergoes transcription but does not encode proteins. These transcription products are referred to as non-protein-coding RNAs (ncRNAs). Various ncRNA transcripts, such as ribosomal RNA, transfer RNA, competitive endogenous RNA (ceRNA), small nuclear RNA (snRNA), and small nuclear RNA (snoRNA), are essential for cellular function. Similarly, a large number of short ncRNAs, such as microRNA (miRNA), endogenous short interfering RNA (siRNA), PIWI-interacting RNA (piRNA), and small nuclear RNA (snoRNA), are also known to play important regulatory roles in eukaryotic cells. Recent studies have demonstrated a group of long ncRNA (lncRNA) transcripts that exhibit cell-type specific expression and are localized to specific intracellular compartments. lncRNAs are also known to play important roles during cell development and differentiation, supporting the view that they were selected during the evolutionary process.

[0122]

[0082] LncRNAs appear to have many different functions. In many cases, they appear to play a role in regulating the activity or localization of proteins or act as a structural framework for intracellular structures. In other cases, lncRNAs are processed to generate multiple small RNAs, or they can regulate how other RNAs are processed. The latest data version generated by the public research consortium GenCode (version 27) cataloged fewer than 16,000 lncRNAs from the human genome, generating nearly 28,000 transcripts; when other databases are included, more than 40,000 lncRNAs are known.

[0123]

[0083] Interestingly, lncRNAs can influence the expression of specific target proteins at specific genomic loci, regulate the activity of protein-binding partners, direct chromatin-modification complexes to their sites of action, and undergo post-transcriptional processing to generate numerous 5'-capped small RNAs. Epigenetic pathways can also regulate the differential expression of lncRNAs.

[0124]

[0084] Increasing evidence also suggests that abnormally expressed lncRNAs play important roles in normal physiological processes as well as in various disease states. LncRNAs are dysregulated in various diseases, including ischemia, heart disease, Alzheimer's disease, psoriasis, and spinocerebellar ataxia type 8. Such dysregulation has also been observed in various types of cancer, e.g., breast cancer, colon cancer, prostate cancer, hepatocellular carcinoma, and leukemia. Several lncRNAs, such as gadd74 and lncRNA-RoR5, regulate cell cycle regulators like cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and p53, thereby providing additional flexibility and robustness to cell cycle progression. Furthermore, some lncRNAs are linked to mitotic processes, such as centrosome satellite RNAs, which are essential for kinetochore formation and are therefore important for chromosome separation during mitosis in humans and flies. Another nuclear lncRNA, MA-lincl, regulates M phase exit by acting as a cis to suppress the expression of the neighboring gene Pura, a regulator of cell proliferation.

[0125]

[0085] lncRNA is a group generally defined as transcripts of 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 lncRNAs as well as shorter transcripts, for example, less than about 200 nt, for example, about 30 to 200 nt.

[0126]

[0086] Accordingly, in some embodiments, delivery of ncRNA, e.g., to a specific brain structure of interest, corrects abnormal RNA expression levels or modulates levels of disease-causing lncRNA. Accordingly, in some embodiments, the present invention provides rAAV, wherein the viral genome is processed to encode therapeutic non-coding RNA (ncRNA). In some embodiments, the ncRNA is a long non-coding RNA (lncRNA) of at least about 200 nucleotides (nt). In some embodiments, the therapeutic agent is an ncRNA of about 25 nt or about 30 nt to about 200 nt in length. In some embodiments, the lncRNA is about 200 nt to about 1,200 nt in length. In some embodiments, the lncRNA is about 200 nt to about 1,100, about 1,000, about 900, about 800, about 700, about 600, about 500, about 400, or about 300 in length.

[0127] C. CRISPR System

[0128]

[0087] Gene editing is a technology that can modify target genes within living cells. Recently, the use of CRISPR’s bacterial immune system to perform on-demand gene editing has revolutionized the way scientists approach genome editing. The Cas9 protein of the CRISPR system, an RNA guide DNA endonuclease, can be modified to target new sites relatively easily by altering the guide RNA sequence. This discovery has made sequence-specific gene editing functionally effective.

[0129]

[0088] Generally, "CRISPR system" refers to transcripts and other elements involved in the expression of or directing the activity of a CRISPR-associated (“Cas”) gene, comprising a sequence encoding the Cas gene collectively, a tracr (trans-activated CRISPR) sequence (e.g., tracrRNA or active fractional tracrRNA), a tracr-mate sequence (a “direct repeat” and a tracrRNA-processed fractional direct repeat in relation to the endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in relation to the endogenous CRISPR system), and / or other sequences and transcripts from a CRISPR locus.

[0130]

[0089] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system may comprise a non-coding RNA molecule (guide) RNA that binds sequence-specifically to DNA and Cas proteins (e.g., Cas9) along with nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system may be, for example, a specific organism containing an endogenous CRISPR system, e.g., Streptococcus pyogenes ( Streptococcus pyogenes It can be derived from a Type I, Type II, or Type III CRISPR system derived from ).

[0131]

[0090] A CRISPR system can induce a disintegration as described herein following a double-strand break (DSB) at a target site. In another embodiment, a Cas9 variant considered as a “nickase” is used to nick a single strand at the target site. By using a paired nickase, specificity directed by a pair of different gRNA targeting sequences can be improved, for example, so that a 5’ overhang is introduced upon the simultaneous introduction of each nick. In another embodiment, a catalytically inactivated Cas9 is fused to a heterogeneous effector domain, such as a transcriptional repressor (e.g., KRAB) or an activator, to influence gene expression. Alternatively, a CRISPR system with a catalytically inactivated Cas9 further comprises a transcriptional repressor or activator fused to a ribosome-binding protein.

[0132]

[0091] In some aspects, a Cas nuclease and gRNA (including a fusion of a target sequence and a crRNA specific to an immobilized tracrRNA) are introduced into a cell. Generally, a target site at the 5' end of the gRNA targets the Cas nuclease to a target site, e.g., a gene, using complementary base pairing. The target site can typically be selected based on the immediate 5' position of a protospacer adjacent motif (PAM) sequence, such as NGG or NAG. In this regard, the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, the CRISPR system is characterized by factors that promote the formation of the CRISPR complex at the site of the target sequence. Typically, the "target sequence" refers to a sequence designed to be complementary to a guide sequence, wherein hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Complete complementarity is not necessarily required, provided that there is sufficient complementarity to induce hybridization and promote the formation of the CRISPR complex.

[0133]

[0092] The target sequence may include any polynucleotide, such as DNA or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of the cell, for example, within organelles of the cell. Generally, a sequence or template that can be used for recombination to a targeted locus containing the target sequence is referred to as an "editing template," "editing polynucleotide," or "editing sequence." In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is homologous recombination.

[0134]

[0093] Typically, in relation to an endogenous CRISPR system, the formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) induces the cleavage of one or both strands at or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs therefrom). A tracr sequence that may include or consist of all or part of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence) may also form part of the CRISPR complex by, for example, hybridizing at least part of the tracr sequence to all or part of a tracr pair sequence operatively linked to the guide sequence. The tracr sequence has sufficient complementarity to the tracr pair sequence involved in hybridization and the formation of the CRISPR complex, for example, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr pair sequence when optimally aligned.

[0135]

[0094] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into cells, and the expression of the CRISPR system elements may direct the formation of a CRISPR complex at one or more target sites. The components may also be delivered to the cell as proteins and / or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr-pair sequence, and the tracr sequence may each be operatively linked to separate regulatory elements on separate vectors. The Cas enzyme may be a target gene under the control of an alternative splicing event regulated as a chimeric target gene microgene or as a target gene for a chimeric microgene transactivator. The gRNA may be under the control of a homeostatic promoter.

[0136]

[0095] Alternatively, two or more elements expressed from the same or different regulatory elements may be combined into a single vector with one or more additional vectors providing any components of the CRISPR system not included in the first vector. The vector may include one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). 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. Where multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different corresponding target sequences within the cell.

[0137]

[0096] The vector may contain a regulatory element operatively linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs thereof or modified versions thereof Includes versions. These enzymes are known; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under approval number Q99ZW2.

[0138]

[0097] CRISPR enzymes are Cas9 (e.g. ,It may be from S. pyogenes or S. pneumonia. The CRISPR enzyme may direct the cleavage of one or both strands at a location in the target sequence, for example, within the target sequence and / or within the complement of the target sequence. The vector may encode a CRISPR enzyme mutated in relation to the corresponding wild-type enzyme, so that the mutated CRISPR enzyme may lack the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nikase (which cleaves a single strand). In some embodiments, the Cas9 nikase may be used in combination with guide sequence(s), for example, two guide sequences that target the sense and antisense strands of a DNA target, respectively. The combination is used to cause niking of both strands and to induce NHEJ or HDR.

[0139]

[0098] In some embodiments, the enzyme-coding sequence encoding the CRISPR enzyme is codon-optimized for expression in specific cells, e.g., eukaryotic cells. Eukaryotic cells may be cells of specific organisms, such as mammals including humans, mice, rats, rabbits, dogs, or non-human primates, or cells derived from them. Generally, codon optimization refers to the process of modifying a nucleic acid sequence for enhanced expression in a host cell of interest by replacing at least one codon of the unique sequence with a codon that is more or possibly more commonly used in the host cell's gene while maintaining the unique amino acid sequence. Various species exhibit specific biases toward specific codons of specific amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the decoding efficiency of messenger RNA (mRNA), which is subsequently thought to depend, above all, on the nature of the codon to be decoded and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNA in a cell generally reflects the codons most commonly used in peptide synthesis. Therefore, genes can be adjusted for optimal gene expression in a given organism based on codon optimization.

[0140]

[0099] Generally, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0141]

[0100] The optimal alignment can be determined using any suitable algorithm for aligning sequences, and non-limiting examples of said algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., 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).

[0142]

[0101] A CRISPR enzyme may be part of a fusion protein comprising one or more heterogeneous protein domains. A CRISPR enzyme fusion protein may comprise any additional protein sequence and any linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, an epitope tag, a reporter gene sequence, and a protein domain having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repressive activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include the histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP), and autofluorescent proteins. CRISPR enzymes may be fused to gene sequences encoding proteins or fragments of proteins that bind to DNA molecules or other cellular molecules, including but not limited to maltose-binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. Additional domains that can form part of a fusion protein containing a CRISPR enzyme are described in US 20110059502, which is incorporated herein by reference.

[0143] D. Therapeutic Proteins

[0144]

[0102] Some embodiments relate to the expression of recombinant proteins and polypeptides. In some aspects, the protein or polypeptide may be modified to increase serum stability. Accordingly, when this application refers to the function or activity of a “modified protein” or a “modified polypeptide,” those skilled in the art will understand that this includes a protein or polypeptide having additional advantages over, for example, an unmodified protein or polypeptide. This is specifically considered to mean that embodiments relating to a “modified protein” may be carried out on a “modified polypeptide” and vice versa.

[0145]

[0103] Recombinant proteins may have deletions and / or substitutions of amino acids; thus, deleted proteins, substituted proteins, and deleted and substituted proteins are modified proteins. In some embodiments, these proteins may additionally include inserted or added amino acids, such as in a fusion protein or a protein having a linker. A “modified deleted protein” may lack one or more residues of the native protein but may retain the specificity and / or activity of the natural protein. A “modified deleted protein” may also have reduced immunogenicity or antigenicity. An example of a modified deleted protein is one in which an amino acid residue is deleted from at least one antigenic region, that is, a region of the protein determined to be antigenic in a specific organism, such as the organism to which the modified protein is administered.

[0146]

[0104] Substitutions or alternative variants typically involve the exchange of one amino acid for another at one or more sites within a protein and may be designed to modify one or more properties of a polypeptide, in particular, its effector function and / or bioavailability. Substitutions may be conserved or non-conserved, that is, one amino acid is replaced with one of a similar form or charge. Conserved substitutions are widely known in the art and include, for example, the following changes: from alanine to serine; from arginine to lysine; from asparagine to glutamine or histidine; from aspartate to glutamate; from cysteine ​​to serine; from glutamine to asparagine; from glutamate to aspartate; from glycine to proline; from histidine to asparagine or glutamine; from isoleucine to leucine or valine; from leucine to valine or isoleucine; from lysine to arginine; and from methionine to leucine or isoleucine. From phenylalanine to tyrosine, leucine, or methionine; from serine to threonine; from threonine to serine; from tryptophan to tyrosine; from tyrosine to tryptophan or phenylalanine; and from valine to isoleucine or leucine.

[0147]

[0105] In addition to deletion or substitution, the modified protein may have a residue insertion, typically involving the addition of at least one residue in the polypeptide. This may involve a targeting peptide or polypeptide, or simply the insertion of a single residue. Terminal additions referred to as fusion proteins are discussed below.

[0148]

[0106] The term “biological and functional equivalent” is well understood in the art and is further defined in detail herein. Accordingly, a sequence comprising about 70% to about 80%, or about 81% to about 90%, or even about 91% to about 99% of amino acids identical or functionally equivalent to the amino acids of the control polypeptide, provided that the biological activity of said protein must be maintained. A recombinant protein may be biologically and functionally equivalent to its intrinsic counterpart under certain circumstances.

[0149]

[0107] This is also understood to be as one of the sequences described herein that satisfy the criteria presented above, wherein the amino acid and nucleic acid sequences may include additional residues, e.g., additional N- or C-terminal amino acids, or 5' or 3' sequences, but still essentially the sequences include the maintenance of biological protein activity related to protein expression. The addition of terminal sequences may include, in particular, various non-coding sequences that flank either the 5' or 3' portion of the coding region, e.g., various internal sequences known to exist within the gene, i.e., introns.

[0150]

[0108] As used herein, the terms protein or peptide generally refer to, but are not limited to, proteins of more than about 200 amino acids up to the full-length sequence decoded from a gene; polypeptides of more 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.

[0151]

[0109] As used herein, “amino acid residue” refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art. In certain embodiments, residues of the protein or peptide are sequential without any non-amino acid interfering with the sequence of the amino acid residues. In other embodiments, the sequence may include one or more non-amino acid moietys. In certain embodiments, the sequence of the protein or peptide residues may be interrupted by one or more non-amino acid moietys.

[0152]

[0110] Accordingly, the term “protein or peptide” encompasses an amino acid sequence containing at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or specific amino acid.

[0153]

[0111] Specific embodiments of the present invention relate to fusion proteins. These molecules may have therapeutic proteins connected to a heterogeneous domain at the N-terminus or C-terminus. For example, the fusion may also enable recombinant expression of the protein in a heterogeneous host using a leader sequence of a different species origin. Another useful fusion preferably involves the addition of a protein affinity tag, e.g., a serum albumin affinity tag or six histidine residues, or an immunologically active domain, e.g., an antibody epitope, to facilitate the purification of the cleavable fusion protein. Non-restriction affinity tags include, but are not limited to, polyhistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST).

[0154]

[0112] Methods for generating fusion proteins are widely known to those skilled in the art. Such proteins can be generated, for example, by de novo synthesis of a complete fusion protein, or by the expression of a complete fusion protein following the attachment of DNA sequences encoding heterogeneous domains.

[0155]

[0113] The generation of a fusion protein that restores the functional activity of the parent protein can be facilitated by linking the gene with a bridging DNA segment encoding a peptide linker that is spliced ​​between parallel polypeptides. The linker is of sufficient length to enable proper folding of the obtained fusion protein.

[0156] III. Method of Administration

[0157]

[0114] In some aspects, viral vectors may be administered directly (in vivo) to a patient, or they may be used to treat cells in vitro or in vitro and subsequently administered to a patient. The term "vector" refers to a small carrier nucleic acid molecule, plasmid, virus (e.g., AAV vector, retroviral vector, lentiviral vector), or other vehicle that can be manipulated by the insertion or incorporation of nucleic acids. A vector, e.g., a viral vector, is used to introduce and / or deliver a nucleic acid sequence to a cell so that the nucleic acid sequence is transcribed therefrom and, if it encodes a protein, subsequently decoded by the cell.

[0158]

[0115] Any suitable cell or mammal may be administered or treated by the methods or uses described herein. Typically, mammals requiring the methods described herein are suspected of having or expressing abnormal or aberrant proteins associated with a disease state. Alternatively, mammalian recipients may have a pathological condition that is compliant with gene replacement therapy. As used herein, “gene replacement therapy” refers to the administration of exogenous genetic material encoding a therapeutic agent to a recipient and subsequent expression of the administered genetic material in the same lineage. Thus, the phrase “pathological condition compliant with gene replacement therapy” encompasses conditions such as genetic diseases (i.e., disease conditions caused by one or more genetic defects), acquired pathologies (i.e., pathological conditions not caused by congenital defects), cancer, and prophylactic processes (i.e., prevention of diseases or undesirable medical conditions). Accordingly, the term “therapeutic agent” as used herein refers to any agent or substance having a beneficial effect on the mammalian recipient. Therefore, "therapeutic agents" encompass both therapeutic and prophylactic molecules having nucleic acid or protein components.

[0159]

[0116] Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), pets (e.g., dogs and cats), farm animals (e.g., horses, cattle, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In certain embodiments, the mammal is a human. In certain embodiments, the mammal is a non-rodent mammal (e.g., humans, pigs, goats, sheep, horses, dogs, etc.). In certain embodiments, the non-rodent mammal is a human. The mammal may be of any age or any developmental stage (e.g., adult, teenager, child, infant, or in-uterine mammal). The mammal may be male or female. In certain embodiments, the mammal may be an animal disease model, e.g., an animal model having or expressing an abnormal or aberrant protein associated with a disease state, or an animal model having insufficient expression of a protein that causes a disease state.

[0160]

[0117] Mammals (subjects) treated with the method or composition described herein include adults (18 years of age or older) and children (under 18 years of age). Adults include the elderly. Representative adults are 50 years of age or older. Children are 1 to 2 years of age, or in age ranges of 2-4, 4-6, 6-18, 8-10, 10-12, 12-15, and 15-18 years. Children also include infants. Infants are typically in age ranges of 1 to 12 months.

[0161]

[0118] In a specific embodiment, the method comprises the step of administering a plurality of viral particles to a mammal as presented herein, such that the severity, frequency, progression, or duration of onset of one or more symptoms of a disease state such as a neurodegenerative disease is reduced, lowered, prevented, suppressed, or delayed. In a specific embodiment, the method comprises the step of administering a plurality of viral particles to a mammal to treat adverse symptoms of a disease state such as a neurodegenerative disease. In a specific embodiment, the method comprises administering a plurality of viral particles to a mammal to stabilize, delay, or prevent the worsening or progression of a disease state such as a neurodegenerative disease, or to reverse or reverse symptoms.

[0162]

[0119] In a specific embodiment, the method delivers a plurality of virus particles to the central nervous system or part thereof of a mammal as presented herein, and the severity, frequency, progression, or onset time of one or more symptoms of a disease state, e.g., a neurodegenerative disease, is reduced, decreased, prevented, inhibited, or delayed by at least about 5 to about 10, about 10 to about 25, about 25 to about 50, or about 50 to about 100 days.

[0163]

[0120] In certain embodiments, symptoms or side effects include early, middle, or late stage symptoms; behavioral, personality, or language symptoms; symptoms of swallowing, movement, seizures, tremors, or restlessness; ataxia; and / or cognitive symptoms such as memory and organizational ability.

[0164] IV. Pharmaceutical Composition

[0165]

[0121] As used herein, the terms “pharmaceuticalally acceptable” and “physiologically acceptable” mean biologically acceptable compositions, formulations, liquids or solids, or mixtures thereof, which are suitable for one or more routes of administration, in vivo delivery, or contact. “Pharmaceutically acceptable” or “physiologically acceptable” compositions are not biologically or otherwise undesirable substances, and, for example, said substances may be administered to a subject without causing substantially undesirable biological effects. The compositions, “pharmaceutically acceptable” and “physiologically acceptable” formulations, and compositions may be sterile. The pharmaceutical formulations and compositions may be used, for example, to administer virus particles to a subject.

[0166]

[0122] The above 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, dispersions and suspension media, coatings, isotonic agents, and absorption promoters or retarders, which are suitable for pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickeners. Supplemental active compounds (e.g., preservatives, antibacterial agents, antiviral agents, and antifungal agents) may also be incorporated into the formulations and compositions.

[0167]

[0123] The pharmaceutical composition typically contains pharmaceutically acceptable excipients. These excipients include any pharmaceutical formulations that can be administered without excessive toxicity and without self-inducing the production of harmful antibodies in the subject receiving the composition. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80, and liquids such as water, saline solution, glycerol, and ethanol. Pharmaceutically acceptable salts may be included, for example, inorganic salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids such as acetate, propionate, malonate, benzoate, etc. Additionally, auxiliary substances such as surfactants, wetting agents or emulsifiers, pH buffers, etc. may be present in this vehicle.

[0168]

[0124] Pharmaceutical compositions may be formulated to be compatible with specific routes of administration or delivery presented herein or known to those skilled in the art. Accordingly, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration or delivery by various routes.

[0169]

[0125] Pharmaceutical forms suitable for injection or infusion of virus particles may include sterile aqueous solutions or dispersions suitable for immediate preparation of sterile injectable or infusionable solutions or dispersions optionally encapsulated in liposomes. In all cases, the final form must be a sterile fluid and stable under conditions of preparation, use, and storage. The liquid carrier or vehicle may be, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Suitable fluidity may be maintained, for example, by the formation of liposomes, by the maintenance of the particle size required in the case of dispersions, or by the use of surfactants. Isotonic agents, for example, sugars, buffers, or salts (e.g., sodium chloride), may be included. Delayed absorption of the injectable composition may be caused by the use of agents that delay absorption in the composition, for example, aluminum monostearate and gelatin.

[0170]

[0126] A solution or suspension of virus particles may optionally contain one or more of the following components: a sterile diluent such as water for injection, a saline solution such as phosphate-buffered saline (PBS), artificial CSF, a surfactant, a fixing oil, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), glycerin, or other synthetic solvents; antimicrobial 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 acetate, citrate, or phosphate, and agents for tonic adjustment such as sodium chloride or dextrose.

[0171]

[0127] Pharmaceutical formulations, compositions, and delivery systems suitable for the compositions, methods, and uses of the present invention are known in the art (see literature: e.g., Remington: The Science and Practice of Pharmacy (2003) 20). th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18 th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12 th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11 th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al. , Drug Delivery Systems (1980), RL Juliano, ed., Oxford, NY, pp. 253-315)).

[0172]

[0128] Viral particles and their compositions may be formulated in the form of dosing units for ease of administration and uniformity of dosage. As used herein, the dosing unit form refers to a physically separated unit fitted to a single dose for an individual to be treated; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect, along with the necessary pharmaceutical carrier. The dosing unit form depends on the number of viral particles deemed necessary to produce the desired effect(s). The required amount may be formulated as a single dose or in multiple dosing units. The dose may be adjusted to a suitable viral particle concentration, optionally combined with an anti-inflammatory agent, and packaged for use.

[0173]

[0129] In one embodiment, the pharmaceutical composition comprises a genetic material sufficient to provide a therapeutically effective amount, that is, an amount sufficient to reduce or improve symptoms or side effects of the disease state in question or an amount sufficient to impart a desired benefit.

[0174]

[0130] As used herein, “unit dosage forms” refer to physically separated units suitable for a single dose for a subject to be treated; each unit contains a predetermined amount optionally combined with a pharmaceutical carrier (excipient, diluent, vehicle, or filler) calculated to produce the desired effect (e.g., prophylactic or therapeutic effect) when administered in one or more doses. Unit dosage forms may be in ampoules and vials, which may include, for example, a liquid composition, or a composition in a lyophilized or lyophilized state; for example, a sterile liquid carrier may be added prior to in vivo administration or delivery. Individual unit dosage forms may be contained in a multi-dose kit or container. Thus, for example, virus particles and their pharmaceutical compositions may be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0175]

[0131] Formulations containing viral particles typically contain an effective amount, which is readily determined by those skilled in the art. Viral particles typically constitute about 1% to about 95% (w / w) of the composition, or may be in a higher range where appropriate. The amount administered depends on factors such as age, body weight, and physical condition of the mammalian or human subject being considered for treatment. The effective dose can be established by those skilled in the art through conventional testing to establish a dose-response curve.

[0176] V. Definition

[0177]

[0132] The terms “polynucleotide,” “nucleic acid,” and “transgenic gene” are used interchangeably herein to refer to all forms of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and oligonucleotides comprising polymers thereof. Polynucleotides include genomic DNA, cDNA and antisense DNA, spliced ​​or unspliced ​​mRNA, rRNA, tRNA, and repressor DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides 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 be of any suitable length. In discussing polynucleotides, the sequence or structure of a specific polynucleotide may be described by providing the sequence in the 5' to 3' direction in accordance with the convention herein.

[0178]

[0133] Nucleic acids encoding polypeptides often contain open read frames encoding the polypeptide. Unless otherwise specified, specific nucleic acid sequences also contain degenerate codon substitutions.

[0179]

[0134] The nucleic acid may comprise one or more expression control or regulatory elements operably linked to an open read frame, wherein one or more regulatory elements are configured to direct the transcription and decoding of a polypeptide encoded by the open read frame in a mammalian cell. Non-limiting examples of expression control / regulatory elements include transcription initiation sequences (e.g., promoters, enhancers, TATA boxes, etc.), decoding 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.

[0180]

[0135] "Promoter" refers to a nucleotide sequence, generally upstream (5') of the coding sequence, that directs and / or controls the expression of the coding sequence by providing recognition to RNA polymerases and other factors necessary for proper transcription. The pol II promoter comprises a minimal promoter, which is a short DNA sequence consisting of a TATA box, and optionally other sequences that serve to specify the transcription initiation site to which regulatory elements are added for expression control. The Type 1 pol III promoter comprises three cis-action sequence elements downstream of the transcription initiation site: a) a 5' sequence element (Block A); b) an intermediate sequence element (Block I); ​​and c) a 3' sequence element (Block C). The Type 2 pol III promoter comprises two essential cis-action sequence elements downstream of the transcription initiation site: a) Box A (5' sequence element); and b) Box B (3' sequence element). Type 3 pol III promoters contain several cis-acting promoter elements upstream of the transcription start site, such as a standard TATA box, proximal sequence element (PSE), and distal sequence element (DSE).

[0181]

[0136] An “enhancer” is a DNA sequence capable of stimulating transcriptional activity and may be an innate element of a promoter or a heterogeneous element that enhances the level of expression or tissue specificity. It may function at any orientation (5’->3’ or 3’->5’) and may function even when located upstream or downstream of the promoter.

[0182]

[0137] The promoter and / or enhancer may be derived entirely from an intrinsic gene, composed of different elements derived from different elements found in nature, or even composed of synthetic DNA fragments. The promoter or enhancer may include a DNA sequence involved in the binding of a protein factor that regulates / controls the effectiveness of transcription initiation in response to stimulation, physiological, or developmental conditions.

[0183]

[0138] Non-limiting examples of promoters include the SV40 early promoter, the mouse mammary tumor virus LTR promoter; the adenovirus major rate promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, e.g., the CMV emediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the pol II promoter, the pol III promoter, synthetic promoters, hybrid promoters, etc. Additionally, sequences derived from non-viral genes, such as the murine metallothionein gene, are also used herein. Exemplary homeostatic promoters include promoters for the following genes encoding specific homeostatic or "housekeeping" functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, actin promoter, U6, and other homeostatic promoters known to those skilled in the art. Additionally, many viral promoters function as homeostasis in eukaryotic cells. These include: early and late promoters of SV40; long-terminal repeats (LTRs) of Moloney's leukemia virus and other retroviruses; and, among many others, the thymidine kinase promoter of herpes simplex virus. Additionally, sequences derived from intron miRNA promoters, such as, for example, miR107, miR206, miR208b, miR548f-2, miR569, miR590, miR566, and miR128 promoters, are used herein (see references: e.g., Monteys et al. , 2010). Therefore, any of the aforementioned homeostatic promoters can be used to control the transcription of heterogeneous gene inserts.

[0184]

[0139] The term "transposable" is used to conveniently refer to nucleic acid sequences / polynucleotides intended for or introduced into a cell or organism. Transposables contain any nucleic acid, such as repressive RNA or genes encoding polypeptides or proteins, and are generally heterogeneous with respect to naturally occurring AAV genome sequences.

[0185]

[0140] The term "transduction" refers to the introduction of a nucleic acid sequence into a cell or host organism via a vector (e.g., a viral particle). Therefore, the introduction of a transgene into a cell by a viral particle may be referred to as the "transduction" of the cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. If the introduced transgene is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it may be stably maintained in the recipient cell or organism and subsequently transferred or inherited by the recipient cell or organism's progeny cells or organisms. Ultimately, the introduced transgene may exist outside the chromosome or merely transiently within the recipient cell or host organism. Thus, a "transduced cell" is a cell into which a transgene has been introduced by transduction. Therefore, a "transduced" cell is a cell into which a transgene has been introduced or its progeny. Transduced cells may proliferate, and the transgene may be transcribed, and the repressor RNA or protein encoded by it may be expressed. Transduced cells for use and methods in gene therapy may exist in mammals.

[0186]

[0141] Transgenic genes under the control of inducible promoters are expressed only in the presence of an inducer or to a greater extent (e.g., transcription under the control of a metallothionein promoter is significantly increased in the presence of a specific metal ion). Inducible promoters contain reactive elements (REs) that stimulate transcription when their inducible factors bind. Examples include REs for serum factors, steroid hormones, retinoic acid, and cyclic AMPs. Promoters containing specific REs can be selected to obtain an inducible response, and in some cases, the RE itself can be attached to other promoters to confer inducibility to the recombinant gene. Thus, by selecting a suitable promoter (constancy versus inducibility, strong versus weak), both the presence and expression level of polypeptide expression in genetically modified cells can be controlled. When a gene encoding a polypeptide is under the control of an inducible promoter, homologous delivery of the polypeptide is triggered by homologous exposure of genetically modified cells to conditions that allow for the transcription of the polypeptide, for example, by the intraperitoneal injection of a specific inducer of the inducible promoter that controls the transcription of the preparation. For example, homologous expression by genetically modified cells of a polypeptide encoded by a gene under the control of a metallothionein promoter is enhanced by contacting the genetically modified cells with a solution containing a homologous appropriate (i.e., inducing) metal ion.

[0187]

[0142] A nucleic acid / transposable is "operably linked" when it is functionally related to another nucleic acid sequence. A nucleic acid / transposable encoding RNAi or a polypeptide, or a nucleic acid directing the expression of a polypeptide, may include an inducible promoter or a tissue-specific promoter to control the transcription of the encoded polypeptide. A nucleic acid operably linked to an expression regulatory element may also be referred to as an expression cassette.

[0188]

[0143] In certain embodiments, CNS-specific or inducible promoters, enhancers, etc. are used in the methods and uses described herein. Non-limiting examples of CNS-specific promoters include those isolated from genes, such as myelin basic protein (MBP), glial fibrinate protein (GFAP), and neuron-specific enolase (NSE). Non-limiting examples of inducible promoters include DNA-responsive elements to ecdysone, tetracycline, hypoxia, and IFN.

[0189]

[0144] In a specific embodiment, the expression control element includes a CMV enhancer. In a specific embodiment, the expression control element includes a beta-actin promoter. In a specific embodiment, the expression control element includes a chicken beta-actin promoter. In a specific embodiment, the expression control element includes a CMV enhancer and a chicken beta-actin promoter.

[0190]

[0145] The terms "modify" or "variant" as used herein and their grammatical variations mean that a nucleic acid, polypeptide, or subsequence thereof deviates from a reference sequence. Accordingly, modified and variant sequences may have substantially the same, greater, or lesser expression, activity, or function as the reference sequence, but may retain at least partial 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, e.g., a missense or nonsense mutation.

[0191]

[0146] "Nucleic acid" or "polynucleotide" variants refer to modified sequences that are genetically altered compared to the wild type. The sequence may be genetically modified without altering the encoded protein sequence. Alternatively, the sequence may be genetically modified to encode a variant protein. Nucleic acid or polynucleotide variants may also refer to codon-modified combination sequences that encode a protein that still retains at least partial sequence identity with respect to a reference sequence, such as the wild-type protein sequence, and also encode a variant variant. For example, some codons of these nucleic acid variants will be changed without altering the amino acids of the protein encoded by them, while some codons of the nucleic acid variants will be changed, which subsequently alters the amino acids of the protein encoded by them.

[0192]

[0147] The terms “protein” and “polypeptide” are used interchangeably herein. The “polypeptide” encoded by the “nucleic acid,” “polynucleotide,” or “transposable gene” described herein comprises a partial or full-length intrinsic sequence, a functional subsequence (fragment) thereof, and sequence variants thereof, as in naturally occurring wild-type and functional polymorphic proteins, insofar as the polypeptide retains some degree of function or activity. Accordingly, in the methods and uses of the present invention, said polypeptide encoded by the nucleic acid sequence does not need to be identical to the endogenous protein that is defective or insufficient, deficient, or absent in activity, function, or expression in treated mammals.

[0193]

[0148] Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (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).

[0194]

[0149] Examples of amino acid modifications are conservative amino acid substitutions or deletions. In certain embodiments, the modified or variant sequence retains at least some of the function or activity of the unmodified sequence (e.g., wild-type sequence).

[0195]

[0150] Another example of amino acid modification is a targeting peptide introduced into the capsid protein of a viral particle. Peptides targeting recombinant viral vectors to the central nervous system, such as to distinct brain regions, have been identified.

[0196]

[0151] Such modified recombinant viruses can preferentially bind to one type of tissue (e.g., CNS tissue) over other types of tissue (e.g., liver tissue). In a specific embodiment, a recombinant virus containing a modified capsid protein can "target" brain vascular epithelial tissue by binding at a higher level than a corresponding unmodified capsid protein. For example, a recombinant virus having a modified capsid protein can bind to brain vascular epithelial tissue at a level 50% to 100% higher than an unmodified recombinant virus.

[0197]

[0152] A “nucleic acid fragment” is a part of a given nucleic acid molecule. In most organisms, deoxyribonucleic acid (DNA) is the genetic material, and ribonucleic acid (RNA) is involved in transferring information contained in DNA to proteins. Fragments and variants of the described nucleotide sequence and proteins or partial-length proteins encoded by them are also included in the invention. “Fragment” or “part” means the full length or less than the full length of a nucleotide sequence or amino acid sequence encoding a polypeptide or protein. In certain embodiments, the fragment or part is biologically functional (i.e., possesses 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the wild-type activity or function).

[0198]

[0153] "Variants" of a molecule are sequences substantially similar to the sequence of a natural molecule. In the case of nucleotide sequences, variants include sequences that encode the same amino acid sequence as the native protein due to the degeneracy of the genetic code. Naturally occurring allelic variants such as these can be identified using molecular biology techniques, for example, polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, for example, those generated using site-directed mutagenesis encoding the native protein, as well as those encoding polypeptides with amino acid substitutions. Generally, the nucleotide sequence variant of the present invention has at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% sequence identity with the intrinsic (endogenous) nucleotide sequence. In certain embodiments, the variant is biologically functional (i.e., possesses 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the wild-type activity or function).

[0199]

[0154] "Conservative variations" of a specific nucleic acid sequence refer to nucleic acids that encode the same or essentially identical amino acid sequence. Due to the degenerate nature of the genetic code, a significant 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 any position where arginine is specified by a codon, the codon can be changed to any corresponding codon described without altering the encoded protein. Such nucleic acid variations are "silent variations," which are a type of "conservatively modified variation." All nucleic acid sequences described herein that encode polypeptides describe all possible silent variations unless otherwise noted. Those skilled in the art will recognize that each codon of a nucleic acid (except ATG, which is generally the only codon for methionine) can be modified by standard techniques to produce a functionally identical molecule. Therefore, each “silent variant” of the nucleic acid encoding the polypeptide is included in each described sequence.

[0200]

[0155] The term "substantial identity" of a polynucleotide sequence means that, when compared to a reference sequence using one of the alignment programs described using standard parameters, the polynucleotide contains 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. Those 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, read frame position, etc. For these purposes, substantial identity of amino acid sequences generally means at least 70%, at least 80%, 90%, or even at least 95% sequence identity.

[0201]

[0156] The term "substantial identity" associated with polypeptides indicates that the polypeptide contains a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79% of sequence identity with a reference sequence over a specific comparison window, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% of sequence identity, or at least 90%, 91%, 92%, 93%, or 94% of sequence identity, or even at least 95%, 96%, 97%, 98%, or 99% of sequence identity. An indication that two polypeptide sequences are identical is that one polypeptide is immunologically reactive with the antibody produced against the second polypeptide. Therefore, the polypeptide is identical to the second polypeptide, for example, when the two peptides differ only by the degree of conservative substitution.

[0202]

[0157] The terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventive measures, wherein the objective is to prevent, inhibit, reduce, or mitigate unintended physiological changes or disorders, such as the onset, progression, or exacerbation of a disorder. For the purposes of the present invention, beneficial or desired clinical outcomes include alleviation of symptoms, reduction in the severity of the disease, stabilization of symptoms or side effects of the disease (i.e., not worsening or progressing), delay or deceleration of disease progression, improvement or alleviation of the disease state, and remission (partial or complete), whether or not they are detected. “Treatment” may also mean extending survival compared to survival expected without treatment. Those requiring treatment include those who already have a pathological condition or disorder and those with a predisposition (e.g., determined by genetic analysis).

[0203] VI. Kit

[0204]

[0158] The present invention provides a packaging material and a kit comprising one or more components therein. The kit generally comprises a label or packaging insert comprising a description of the component or instructions for the use of the component in vitro, in vivo, or in vitro. The kit may comprise said components, for example, nucleic acids, recombinant vectors, and / or an assembly of virus particles.

[0205]

[0159] A kit refers to a physical structure containing one or more components of a kit. The packaging material can maintain the components in a sterile state and may consist of materials commonly used for this purpose (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0206]

[0160] The label or insert may contain clinical pharmacology, pharmacokinetics, and pharmacodynamics of the active ingredient(s), including identification information for one or more components therein, dosage, and mechanism of action. The label or insert may contain information identifying the manufacturer, lot number, manufacturing location and date, and expiration date. The label or insert may contain information identifying the manufacturer information, lot number, manufacturer location and date. The label or insert may contain information regarding the disease for which the kit components may be used. The label or insert may contain instructions for a clinician or subject to use one or more of the kit components in a method, use, or treatment protocol, or therapeutic use. The instructions may include dosage, frequency, or duration, and instructions for implementing any of the method, use, treatment protocol, or prophylactic or therapeutic use.

[0207]

[0161] Labels or inserts may contain information regarding any benefit that the ingredient may provide, such as prophylactic or therapeutic benefits. Labels or inserts may contain information regarding potential side effects, complications, or reactions, such as warning the subject or clinician about situations where the use of a particular composition is inappropriate. Side effects or complications may occur if the subject has taken or is currently taking one or more other drugs that are incompatible with the composition, or if the subject has received, is scheduled to receive, or is currently receiving another therapeutic protocol or therapeutic regimen, and thus the guidance may include information regarding such incompatibility.

[0208]

[0162] The cover or insert may separately include "printed material" such as paper or cardboard, or be fixed to the component, kit, or packaging material (e.g., box), or attached to an ampoule, tube, or vial containing the kit component. The cover or insert may further include a computer-readable medium such as a cover printed with a barcode, a disc, an optical disc such as a CD- or DVD-ROM / RAM, a DVD, an MP3, or a magnetic / optical storage medium such as RAM and ROM, a flash memory, a hybrid of such as a memory type card, and a hybrid of such things.

[0209] VII. Examples

[0210]

[0163] The following examples are incorporated to demonstrate preferred embodiments of the present invention. Those skilled in the art will understand that the techniques described in the following examples represent techniques discovered by the inventors to effectively carry out the present invention and may be considered to constitute a preferred mode of carrying out the invention. However, those skilled in the art will understand, by taking into account the contents of this specification, that similar or similar results can be obtained by making various changes to the specific embodiments disclosed without departing from the concept and scope of the present invention.

[0211] Example 1 - Identification of AAV variants targeting brain parenchyma

[0212]

[0164] Advanced barcode AAV libraries were developed using AAV1, AAV2, and AAV9 capsids as starting platforms. AAV1, AAV2, and AAV9 peptide display libraries were generated by inserting random sequences at position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, and position 588 of the AAV9 capsid, respectively (Fig. 1). The libraries were various 1 x 10 7 It had a unique clone of (Fig. 3).

[0213]

[0165] To test the utility of the library, a pilot study was conducted using bench-grade (low titer, low purity) capsid variant AAV2. The AAV2 library was administered at 8 x 10⁶ per animal. 10 The vector genome was intravenously injected into two C57BL / 6 mice. After 72 hours, the cerebral cortex, cerebellum, and spinal cord were dissected. For reference, the heart, skeletal muscle, and diaphragm were collected separately to identify myorticity. Viral genomic DNA was isolated, and the recovered random oligonucleotide sequences were amplified by PCR. PCR products from the brain were pooled to generate a 2-round library, which was then administered to two mice at a rate of 4 x 10⁶ per animal. 10 The vector genome was injected. After the second injection, the vector genome was retrieved as before and applied to NexGen sequencing along with the starting library and first-round tissues. To test whether sequences appearing abundant in brain tissue could actually be extended for AAV2 to reach the brain, individual hits were cloned into an AAV2 capsid packaging plasmid to generate eGFP-expressing AAV2. A bench-grade vector was constructed, and 3 x 10 of the AAV2-based capsid variant virus 10 The vector genome was injected into mice. After 4 weeks, eGFP fluorescence was observed in the brain even for these low-titer variants.

[0214]

[0166] Using these advanced barcode AAV libraries, AAV variants capable of targeting apparent primate brain structures in non-human primates were identified. The AAV1, AAV2, and AAV9 libraries were delivered to a single non-human primate via intraventricular injection (Fig. 2). 72 hours after injection, brain regions were microdissected for viral DNA isolation and AAV DNA amplified by PCR. The products were pooled and used to package a second-round library to be further injected into NHPs. The brain regions were subsequently microdissected 12 days after injection. After the second round of panning, the vector genomes were recovered and applied for next-generation sequencing. Specifically, genomic DNA extracted from Round 1 and Round 2 tissues was PCR amplified to generate Illumina Amplicon sequencing libraries at the locations of the vector barcodes. The obtained libraries were pooled and developed on a single lane of an Illumina HiSeq 4000 using 100 bp single-terminal read chemistry. To illustrate the utility of the approach, various target regions such as the ependymal, meninges, and cerebellum were tested as examples. In general, the sequences directing AAVx to the ependymal, meninges, and cerebellum were different and varied depending on the different serotypes.

[0215]

[0167] Round-by-round enrichment graphs (Fig. 4) and heatmaps (Figs. 5 and 6) were generated for the following tissues: brainstem, caudate, cerebellar cortex (Fig. 5), cerebral cortex, ependymoma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, and thalamus. These show the enrichment of the barcodes indicated at baseline (Round 0) and describe the biological pathways passing through the rhesus macaque after Rounds 1 and 2. To generate these, fastq result files for each tissue and round combination were processed using a custom Python script designed to extract and quantify unique barcode configurations observed at the DNA level. A custom R script was used to calculate the percentage of barcodes present in each sample and to convert DNA barcodes to amino acid barcodes. Table 1 corresponds to samples processed with an AAV1-derived library; Table 2 represents tissues processed with an AAV2-derived library; and Table 3 corresponds to samples processed with an AAV9-derived library. Top hits were selected from these three libraries and assembled and generated into a validation library containing 50 (AAV1), 58 (AAV2), and 30 (AAV9) derived barcodes. This validation library was delivered to additional Rhesus macaques via ICV injection. The tissues were re-collected and processed to facilitate the recovery of barcode abundance by deep sequencing. Barcode abundance was evaluated in the recovered tissues and the input virus library. The enrichment value for each barcode was calculated relative to its abundance in the input virus library. The obtained relative enrichment values ​​are a strong indicator of vector performance among the various tissues evaluated and facilitate the identification of a wide range of specific AAV vector variants (Figs. 7a-c).

[0216]

[0168] To verify the specificity of the identified cell types, AAV9-1999 (containing the targeting peptide sequence of KGGGFHG; Sequence No.: 110) was selected for in vivo verification. An eGFP expression construct was packaged into AAV9-1999 driven by a CAG promoter. 1.5E13 vg of AAV9-1999 was administered to the left ventricle of 5-year-old female Rhesus macaques via ICV injection. Brains were collected 30 days after injection for histological analysis. Cerebellar slices were H&E stained to depict the transduction pattern of AAV9-1999 (Fig. 8). Cochleas were also collected from the animals and surprisingly showed potent transduction of hair cells. Additionally, AAV9-1999 and AAV9 capsids containing the eGFP construct were delivered to C57BL / 6 p0 mouse pups by ICV injection at 1E10 vg per hemisphere. After 21 days, the mice were perfused. The whole loaded brain (Fig. 9a), a 40 μm forebrain thalamic slice (Fig. 9b), a 40 μm S1 cortical slice (Fig. 9c, left), a 40 μm hippocampal slice (Fig. 9c, middle), a 40 μm cerebellar thalamic slice (Fig. 9c, right), and a 40 μm lumbar spinal cord coronal slice (Fig. 9d) were imaged for eGFP fluorescence signals. AAV9-1999 injected into Bl / 6 neonatal mouse pups showed greater ubiquitous expression than dose-matched injection of AAV9.

[0217]

[0169] A mixture of four modified AAVs was injected into a single adult Rhesus macaque: AAV9 with the RGDLQWV (SEQ No. 113) targeting peptide sequence and the mTAGBFP2 tag; AAV1 with the ERDRTRG (SEQ No. 21) targeting peptide sequence as the mTFP1 tag; AAV2 with the GRGAPGG (SEQ No. 80) targeting peptide sequence and the mNG tag; and AAV2 with the DDPSARR (SEQ No. 53) targeting peptide sequence and the mRuby3 tag. The viruses were directly mixed in equal volumes to achieve the final total dose for each as follows:

[0218] AAV9.RGDL mTagBFP2 6.13E12 Total vg

[0219] AAV1.ERDR mTFP1 1.23E13 total vg

[0220] AAV2.GRGA mNG 8.8E12 total vg

[0221] AAV2.DDPS mRuby3 1.32E13 Total vg

[0222]

[0170] The brain was collected on day 30 after injection for fluorescence imaging. Lateral ventricular sections (Fig. 10a), fourth ventricular sections (Fig. 10b), and meningeal sections (Fig. 10c) were imaged for mTagBFP2, mTFP2, mNG, and mRuby3 fluorescence signals.

[0223]

[0171] Further experiments were conducted by injecting AAV9-1999 into the cochlea of ​​*Rhesus macaque*. Based on the results of cochlear transduction, animals were administered AAV9-1999 into the lateral ventricles. A single animal was administered 3E11 vg of AAV9-1999 via direct injection into the round window with a canal opening (Fig. 11a-c).

[0224] * * *

[0225]

[0172] All methods disclosed and claimed herein can be made and performed without excessive experimentation in consideration of the contents of this specification. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that various modifications can be applied to the methods, steps of said methods, or the order of said steps of said methods without departing from the concept, meaning, and scope of the present invention as understood in the art. More specifically, it will be apparent that certain formulations that are both chemically and physiologically related may replace the formulations described herein if the same or similar results are achieved by using them. All such similar substitutions and modifications apparent to those skilled in the art are deemed to fall within the meaning, scope, and concept of the present invention as defined in the appended claims.

[0226] References

[0227] The following references are incorporated herein by reference in detail to the extent that they provide exemplary procedures for the contents described herein or other detailed explanations to supplement them.

[0228]

[0229]

Claims

Claim 1 A modified AAV capsid protein comprising a viral vector containing a modified adeno-associated virus (AAV) capsid protein, the modified AAV capsid protein comprising a targeting peptide that targets a unique brain structure, wherein the targeting peptide comprises SEQ ID NO. 110 and is a sequence of up to 10 amino acids in length, and the unique brain structure is selected from the group consisting of the brainstem, caudate, cerebellar cortex, cerebral cortex, ependymal space, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, and thalamus. Claim 2 The modified AAV capsid protein of claim 1, wherein the modified AAV9 capsid protein is derived from an AAV9 capsid protein having the amino acid sequence of SEQ ID NO. 140, and the modified AAV capsid protein in which the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. Claim 3 A modified AAV capsid protein according to paragraph 2, wherein the targeting peptide is flanked by a linker sequence, and the linker sequence on each side of the targeting peptide is 2 or 3 amino acids in length. Claim 4 A modified AAV capsid protein according to paragraph 3, 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. Claim 5 In claim 4, the modified AAV9 capsid protein has a sequence that is at least 95% identical to SEQ ID NO. 143, a modified AAV capsid protein. Claim 6 In claim 5, the modified AAV capsid protein in which the targeting peptide is located at amino acid residues 592 to 598 of SEQ ID NO.

143. Claim 7 In paragraph 2, the targeting peptide is a modified AAV capsid protein having a length of 7 amino acids and consisting of SEQ ID NO.

110. Claim 8 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is a brainstem and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 9 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is unknown and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 10 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is the cerebellar cortex and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 11 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is a cerebral cortex and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 12 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is an ependymal membrane and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 13 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is a globus pallidus and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 14 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is the hippocampus and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 15 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is a meninges and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 16 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is an optic nerve and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 17 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is a cutaneous structure and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 18 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is a spinal cord and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 19 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is the substantia nigra and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 20 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is the subthalamic nucleus and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 21 A modified AAV capsid protein according to claim 1, wherein the unique brain structure is the thalamus and the modified AAV capsid protein is a modified AAV9 capsid protein. Claim 22 A nucleic acid comprising a sequence encoding a modified capsid protein of any one of claims 1 to 21. Claim 23 A recombinant adeno-associated virus (rAAV) virus comprising a modified capsid protein of any one of claims 1 to 21. Claim 24 A viral vector comprising a nucleic acid encoding a modified capsid protein of any one of claims 1 to 21. Claim 25 A virus vector according to paragraph 24, further comprising a nucleic acid sequence encoding the nucleic acid of interest. Claim 26 A viral vector comprising a nucleic acid encoding a modified capsid protein of any one of claims 1 to 21; or an in vitro cell comprising a viral vector comprising a nucleic acid encoding a modified capsid protein of any one of claims 1 to 21 and a nucleic acid sequence encoding a nucleic acid of interest. Claim 27 A pharmaceutical composition for use in treating a neurodegenerative disease in mammals, comprising: a recombinant adeno-associated virus (rAAV) virus comprising a modified capsid protein of any one of claims 1 to 21; a viral vector comprising a nucleic acid encoding a modified capsid protein of any one of claims 1 to 21; or a viral vector comprising a nucleic acid encoding a modified capsid protein of any one of claims 1 to 21 and a nucleic acid sequence encoding a nucleic acid of interest, and a pharmaceutically acceptable carrier. Claim 28 A pharmaceutical composition according to claim 27, wherein the neurodegenerative disease is Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, Alzheimer's disease, polyglutamine recurrent disease, or Parkinson's disease. Claim 29 A pharmaceutical composition in which the mammal of claim 27 is a human. 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