Adeno-Associated Virus Vector Targeting the Cerebral Microvasculature

Abstract

Provided herein are targeting peptides that deliver a drug to the cerebral microvasculature, and vectors containing sequences encoding the targeting peptides.

Description

Technical Field

[0001] Reference to Related Applications This application claims the benefit of priority based on U.S. Provisional Application No. 63 / 341,664, filed on May 13, 2022, the entire content of which is incorporated herein by reference.

[0002] Reference to Sequence Listing This application contains an electronically submitted Sequence Listing XML, which is incorporated herein by reference in its entirety. The Sequence Listing XML created on May 9, 2023, is named CHOPP0055WO_ST26.xml and is 55,520 bytes in size.

[0003] 1. Field The present disclosure generally relates to the fields of medicine and virology. More specifically, it relates to compositions and methods for delivering molecular therapeutics to the cerebral microvasculature.

Background Art

[0004] 2. Description of Related Art Various strategies for generating AAV vector variants have been developed, including rational design and directed evolution. Rational design approaches utilize knowledge of the AAV capsid to add target-specific changes to the capsid to alter the efficiency or specificity of transduction, for example, adding tyrosine mutations on the capsid surface to increase transduction efficiency. The cerebral microvasculature is a major part of the blood-brain barrier (BBB) and an important target for gene therapy. rAAV targeted to the cerebral microvasculature has the potential to treat diseases that affect the function of the BBB. However, there are no AAV variants that specifically or efficiently target the cerebral microvasculature. Therefore, there is a need for AAV variants that can target the cerebral microvasculature.

Summary of the Invention

[0005] Summary Provided herein are viral vectors each comprising a modified capsid, wherein the modified capsid comprises at least one amino acid sequence that targets the viral vector to the cerebral microvasculature, such as brain endothelial cells.

[0006] In one aspect, provided is a modified AAV capsid protein comprising a targeting peptide that targets a viral vector comprising the modified AAV capsid protein to the cerebral microvasculature, wherein the targeting peptide is 3 to 10 amino acids in length. In some aspects, the modified AAV capsid protein has a sequence of any one of SEQ ID NO: 26, 27, 25, and 28-42. 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.

[0007] In some aspects, the modified AAV capsid protein is derived from the AAV1 capsid protein (see SEQ ID NO: 1), and the targeting peptide is inserted after residue 590 of the AAV1 capsid protein. In some aspects, a linker sequence is adjacent to the targeting peptide, and the linker sequences on each side of the targeting peptide are 2 or 3 amino acids in length. In some aspects, the linker sequence on the N-terminal side of the targeting peptide is SSA, and the linker sequence on the C-terminal side of the targeting peptide is AS. In some aspects, the modified AAV1 capsid protein has a sequence that is at least 95% identical to SEQ ID NO: 4. In some aspects, the targeting peptide has a sequence provided in the examples. In some aspects, the targeting peptide has a sequence of any one of SEQ ID NO: 7-12. In some aspects, the modified AAV1 capsid protein has a sequence of any one of SEQ ID NO: 25-30.

[0008] In some embodiments, the modified AAV capsid protein is derived from the AAV2 capsid protein (see SEQ ID NO:2), and the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. In some embodiments, the targeting peptide is flanked by linker sequences, and the linker sequences on each side of the targeting peptide are 2 or 3 amino acids in length. In some embodiments, the linker sequence on the N-terminal side of the targeting peptide is AAA, and the linker sequence on the C-terminal side of the targeting peptide is AA. In some embodiments, the modified AAV2 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:5. In some embodiments, the targeting peptide has a sequence provided in the Examples. In some embodiments, the targeting peptide has any one of the sequences of SEQ ID NOs: 13-15. In some embodiments, the modified AAV2 capsid protein has any one of the sequences of SEQ ID NOs: 31-33.

[0009] In some embodiments, the modified AAV capsid protein is derived from the AAV9 capsid protein (see SEQ ID NO:3), and the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. In some embodiments, the targeting peptide is flanked by linker sequences, and the linker sequences on each side of the targeting peptide are 2 or 3 amino acids in length. In some embodiments, the linker sequence on the N-terminal side of the targeting peptide is AAA, and the linker sequence on the C-terminal side of the targeting peptide is AS. In some embodiments, the modified AAV9 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:6. In some embodiments, the targeting peptide has a sequence provided in the Examples. In some embodiments, the targeting peptide has any one of the sequences of SEQ ID NOs: 16-26. In some embodiments, the modified AAV9 capsid protein has any one of the sequences of SEQ ID NOs: 34-42.

[0010] In some aspects, the targeting peptide has the sequences provided in the Examples. In some aspects, the target peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-26, or a sequence of up to 10 amino acids in length having within it an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-26. In some aspects, the targeting peptide is 7 amino acids in length and has within it an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-26, or an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-26. In some aspects, the modified AAV capsid protein has any one of the sequences of SEQ ID NOs: 25-42.

[0011] In one aspect, provided herein is a nucleic acid comprising a sequence encoding any modified capsid protein of an aspect of the invention.

[0012] In one aspect, provided herein is a recombinant adeno-associated virus (rAAV) comprising any modified capsid protein of an aspect of the invention. In some aspects, combinations of rAAVs are provided.

[0013] In one aspect, provided herein is a viral vector comprising a nucleic acid encoding any modified capsid protein of an aspect of the 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.

[0014] In one aspect, provided herein is a cell comprising any viral vector of an aspect of the invention. In some aspects, the cell is a mammalian cell, such as a human cell. In some aspects, the cell is in vitro or in vivo.

[0015] In one aspect, provided herein is a pharmaceutical composition comprising a viral vector of an aspect of the invention and a pharmaceutically acceptable carrier.

[0016] In one aspect, provided herein is a method of delivering a drug to a brain endothelial cell of a subject, comprising administering a virus of an aspect of the invention to the subject.

[0017] In some aspects, the drug is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, a therapeutic protein, or a CRISPR system. In some aspects, the administration is administration to the central nervous system. In some aspects, the administration is administration into the cisterna magna, the intracerebroventricular space, the mantle, the ventricle, the subarachnoid space, and / or the intrathecal space. In some aspects, the ventricle is the rostral lateral ventricle, and / or the caudal lateral ventricle, and / or the right ventricle, and / or the left ventricle, and / or the right rostral lateral ventricle, and / or the left rostral lateral ventricle, and / or the right caudal lateral ventricle, and / or the left caudal lateral ventricle. In some aspects, the administration is systemic administration.

[0018] In some aspects, multiple viral particles are administered. In some aspects, the virus is administered at a dose of about 1×10 6 ~about 1×10 18 vector genomes (vg / kg) per kilogram. In some aspects, the virus is about 1×10 7 ~1×10 17 、about 1×10 8 ~1×10 16 、about 1×10 9 ~1×10 15 、about 1×10 10 ~1×10 14 、about 1×10 10 ~1×10 13 、about 1×10 10 ~1×10 13 、about 1×10 10 ~1×10 11 、about 1×1011 ~1×10 12 、 about 1×10 12 ~×10 13 、 or about 1×10 13 ~1×10 14 and is administered at a dose of ~1×10 vg / kg. In some instances, the subject is a human.

[0019] In one aspect, provided herein is a method of treating a mammalian disease, comprising administering a virus of an aspect of the invention to a mammal. In some instances, the disease is a disease affecting the central nervous system (CNS), such as a lysosomal storage disorder, Glut1 deficiency, or multiple sclerosis. In some instances, the mammal is a human.

[0020] Other objects, features, and advantages of the invention will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description, it is to be understood that this detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only and are not intended to be limiting.

Brief Description of the Drawings

[0021] The following drawings form a part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0022]

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Mode for Carrying Out the Invention

[0023] Detailed Description The brain microvasculature is a major part of the blood - brain barrier (BBB) and an important target for gene therapy. rAAV targeted to the brain microvasculature has the potential to treat diseases that affect the function of the BBB (e.g., Glut1 deficiency or multiple sclerosis). Systemically delivered rAAV targeting the brain microvasculature also presents an opportunity to deliver secreted proteins across the BBB without invasive surgery. This is a promising approach for lysosomal storage diseases (e.g., CLN2 disease, mucopolysaccharidosis IIIA, etc.) as well as for Alzheimer's disease. rAAV targeting the brain microvasculature has been used to treat mouse models of central nervous system diseases, such as lysosomal storage diseases. However, there is currently no AAV vector that can efficiently target the brain microvasculature in primates. Provided herein are AAV capsids that can robustly transduce the brain microvasculature. These AAV capsids can be used to package a variety of therapeutic payloads.

[0024] Provided herein are viral vectors each comprising a modified capsid, where the modified capsid comprises at least one amino acid sequence that targets the viral vector to splenocytes in the brain. In certain embodiments, the AAV is AAV1, AAV2, or AAV9. Exemplary wild-type reference AAV1 capsid protein sequences are provided in SEQ ID NO:1. Exemplary wild-type reference AAV2 capsid protein sequences are provided in SEQ ID NO:2. Exemplary wild-type reference AAV9 capsid protein sequences are provided in SEQ ID NO:3. In certain aspects, the targeting peptide is inserted at position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, or position 588 of the AAV9 capsid. Exemplary modified AAV1 capsid protein sequences are provided in SEQ ID NO:4, SSAX 7 AS (the first SSA and the last AS are linker sequences, and X 7 represents the targeting peptide), indicating the insertion of the targeting peptide after position 590. Exemplary modified AAV2 capsid protein sequences are provided in SEQ ID NO:5, AAAX 7 AA (the first AAA and the last AA are linker sequences, and X 7 represents the targeting peptide), indicating the insertion of the targeting peptide after position 587. Exemplary modified AAV9 capsid protein sequences are provided in SEQ ID NO:6, AAAX 7 AS (the first AAA and the last AS are linker sequences, and X 7 represents the targeting peptide), indicating the insertion of the targeting peptide after position 588.

[0025] I. Adeno-Associated Virus (AAV) Vectors Adeno-associated virus (AAV) is a small non-pathogenic virus of the Parvoviridae family. To date, a number of serologically distinct AAVs have been identified, with more than 12 isolated from humans or primates. AAV is distinguished from other members of this family by its dependence on a helper virus for replication.

[0026] The AAV genome can exist episomally without integrating into the host cell genome; has a broad host range; can transduce both dividing and non-dividing cells in vitro and in vivo, and maintain high-level expression of the transduced gene. AAV virus particles are thermostable; resistant to solvents, detergents, pH changes, and temperature; and can be column-purified and / or concentrated by CsCl gradient or other means. The AAV genome contains single-stranded deoxyribonucleic acid (ssDNA) of either the plus or minus strand. The approximately 4.7 kb AAV genome consists of one segment of single-stranded DNA of either plus or minus polarity. The ends of the genome are short inverted terminal repeats (ITRs) that fold into hairpin structures and can function as origins of viral DNA replication.

[0027] The AAV "genome" ultimately refers to the recombinant nucleic acid sequence that is finally packaged or encapsulated to form AAV particles. AAV particles often contain an AAV genome packaged by AAV capsid proteins. When a recombinant plasmid is used to construct or produce a recombinant vector, the AAV vector genome does not contain the "plasmid" portion that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone" and is important for plasmid cloning and amplification, which are necessary processes for plasmid propagation and production, but which are not themselves packaged or encapsulated into virus particles. Thus, the AAV vector "genome" refers to the nucleic acid packaged or encapsulated by AAV capsid proteins.

[0028] An AAV virion (particle) is a non-enveloped icosahedral particle with a diameter of approximately 25 nm that contains an AAV capsid. The AAV particles contain icosahedral symmetry composed of three related capsid proteins, VP1, VP2, and VP3, which interact with each other to form the capsid. Most native AAV genomes often contain two open reading frames (ORFs), which may also be referred to as the left ORF and the right ORF. The right ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins are often found in a ratio of 1:1:10, respectively, although different ratios may occur, and all are derived from the right ORF. The capsid proteins VP1, VP2, and VP3 differ from each other by alternative splicing and the use of rare start codons. Deletion analysis has shown that the removal or alteration of VP1 translated from a message undergoing alternative splicing results in a decrease in the yield of infectious particles. Mutations within the VP3 coding region result in the failure to produce single-stranded progeny DNA or infectious particles. In certain embodiments, the genome of the AAV particle encodes one, two, or all three of the VP1 polypeptide, VP2 polypeptide, and VP3 polypeptide.

[0029] The left ORF often encodes non-structural Rep proteins, Rep 40, Rep 52, Rep 68, and Rep 78, which are involved in the control of replication and transcription in addition to the production of single-stranded progeny genomes. Two of the Rep proteins have been associated with the preferential integration of the AAV genome into the region of the q arm of human chromosome 19. Rep68 / 78 has been shown to have NTP-binding activity and also DNA helicase and RNA helicase activities. Some of the Rep proteins have a nuclear localization signal and also some potential phosphorylation sites. In certain embodiments, the genome of AAV (e.g., rAAV) encodes some or all of the Rep proteins. In certain embodiments, the genome of AAV (e.g., rAAV) does not encode Rep proteins. In certain embodiments, one or more of the Rep proteins may be delivered in trans and thus are not included in AAV particles containing a nucleic acid encoding the polypeptide.

[0030] The ends of the AAV genome contain short inverted terminal repeats (ITRs) that have the potential to fold into a T-shaped hairpin structure that functions as the origin of viral DNA replication. Thus, the genome of AAV contains one or more (e.g., a pair of) ITR sequences adjacent to the single-stranded viral DNA genome. The ITR sequences often each have a length of about 145 bases. Within the ITR region, two elements, the GAGC repeat motif and the terminal resolution site (trs), which are thought to be central to the function of the ITR, are described. The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation. This binding positions Rep68 / 78 for cleavage at the trs, which occurs site-specifically and strand-specifically. In addition to their role in replication, these two elements also appear to be central to viral integration. The integration locus on chromosome 19 contains a Rep-binding site adjacent to the trs. These elements have been shown to be functional and necessary for locus-specific integration.

[0031] The term "recombinant" is generally used to mean that a composition has been engineered (i.e., modified) in a manner that does not occur naturally, either as a modifier of a vector such as a recombinant viral vector, e.g., a recombinant lentiviral vector or a recombinant parvovirus (e.g., AAV) vector, or as a modifier of a sequence such as a recombinant nucleic acid sequence and a recombinant polypeptide. Specific examples of recombinant vectors, e.g., recombinant AAV vectors, recombinant retroviral vectors, or recombinant lentiviral vectors, are those in which a nucleic acid sequence that is not normally present in the wild-type viral genome has been inserted into the viral genome. Examples of recombinant nucleic acid sequences are those in which a nucleic acid (e.g., a gene) encodes an inhibitory RNA that has been cloned into a vector and that may or may not contain the 5' region, 3' region, and / or intron regions normally associated with the gene within the viral genome. The term "recombinant" is not necessarily always used herein with respect to vectors such as viral vectors and sequences such as polynucleotides, but notwithstanding such omissions, "recombinant" forms that include nucleic acid sequences, polynucleotides, transgenes, etc. are clearly included.

[0032] A recombinant viral "vector" is obtained from a wild-type viral genome by using molecular methods to remove a portion of the wild-type genome from the virus and replace it with a non-native nucleic acid, e.g., a nucleic acid sequence. Typically, e.g., in the case of AAV, one or both of the terminal inverted repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. A "recombinant" viral vector (e.g., rAAV) is distinguished from a viral (e.g., AAV) genome because a portion of the viral genome has been exchanged for a non-native sequence, e.g., a nucleic acid encoding a transactivator, or a nucleic acid encoding an inhibitory RNA, or a nucleic acid encoding a therapeutic protein, with respect to the viral genomic nucleic acid. Thus, the incorporation of such non-native nucleic acid sequences defines the viral vector as a "recombinant" vector, and a "recombinant" vector is also referred to as an "rAAV vector" in the case of AAV.

[0033]

[0033] In certain embodiments, an AAV (e.g., rAAV) contains two ITRs. In certain embodiments, an AAV (e.g., rAAV) contains a pair of ITRs. In certain embodiments, an AAV (e.g., rAAV) contains a pair of ITRs adjacent to (i.e., at each of its 5' and 3' ends) a nucleic acid sequence encoding at least a polypeptide having a function or activity.

[0034] AAV vectors (e.g., rAAV vectors) can be packaged for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo, and are referred to herein as "AAV particles." When a recombinant AAV vector is encapsulated or packaged in an AAV particle, the particle is also referred to as an "rAAV particle." In certain embodiments, the AAV particle is an rAAV particle. rAAV particles often contain an rAAV vector or a portion thereof. An rAAV particle can be one or more rAAV particles (e.g., a number of AAV particles). rAAV particles typically contain a protein (e.g., a capsid protein) that encapsulates or packages the rAAV vector genome. Note that reference to an rAAV vector can also be used to refer to an rAAV particle.

[0035] Any suitable AAV particle (e.g., rAAV particle) can be used for the methods or uses herein. The rAAV particle, and / or the genome contained therein, can be derived from any suitable serotype or strain of AAV. The rAAV particle, and / or the genome contained therein, may be derived from two or more serotypes or strains of AAV. Thus, rAAV can contain the protein and / or nucleic acid of any serotype or strain of AAV or a portion thereof, where the AAV particle is suitable for infection and / or transduction of mammalian cells. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, and AAV-2i8.

[0036] In certain embodiments, a plurality of rAAV particles comprises particles of the same strain or serotype (or subgroup or variant), or particles derived therefrom. In certain embodiments, a plurality of rAAV particles comprises a mixture of two or more different rAAV particles (e.g., of different serotypes and / or strains).

[0037] As used herein, the term "serotype" is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such differences in cross-reactivity are generally due to differences in capsid protein sequences / epitopes (e.g., differences in the sequences of VP1, VP2, and / or VP3 of an AAV serotype). An AAV variant that includes a capsid variant may not be serologically distinct from a reference AAV or other AAV serotype, but has at least one nucleotide or amino acid residue that is different as compared to the reference or other AAV serotype.

[0038] In certain embodiments, an rAAV vector based on the genome of a first serotype corresponds to one or more of the serotypes of the capsid proteins that package the vector. For example, the serotype of one or more AAV nucleic acids (e.g., ITRs) that make up the AAV vector genome corresponds to the serotype of the capsid that makes up the rAAV particle.

[0039] In certain embodiments, the rAAV vector genome may be based on the genome of an AAV (e.g., AAV2) serotype that is distinct from one or more of the serotypes of AAV capsid proteins that package the vector. For example, at least one or more of the three capsid proteins may be derived from a different serotype, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype, or variants thereof, while the rAAV vector genome may contain nucleic acids (e.g., ITRs) derived from AAV2.

[0040] In certain embodiments, the rAAV particles or its vector genome with respect to a reference serotype has a polynucleotide, polypeptide, or subsequence that comprises, or consists of, a sequence that is at least 60% or more identical (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) to a polynucleotide, polypeptide, or subsequence of particles of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8. In a specific embodiment, the rAAV particles or its vector genome with respect to a reference serotype has a capsid or ITR sequence that comprises, or consists of, a sequence that is at least 60% or more identical (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) to a capsid or ITR sequence of the serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8.

[0041] In certain embodiments, the methods herein include the use, administration, or delivery of particles of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74, or rAAV-2i8.

[0042] In certain embodiments, the methods herein include the use, administration, or delivery of rAAV2 particles. In certain embodiments, the rAAV2 particles comprise an AAV2 capsid. In certain embodiments, the rAAV2 particles are at least 60%, 65%, 70%, 75%, or more identical to the corresponding capsid proteins of native or wild-type AAV2 particles, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical, and include one or more capsid proteins (e.g., VP1, VP2, and / or VP3). In certain embodiments, the rAAV2 particles are at least 75% or more identical to the corresponding capsid proteins of native or wild-type AAV2 particles, e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical, and include capsid proteins VP1, VP2, and VP3. In certain embodiments, the rAAV2 particles are variants of native or wild-type AAV2 particles. In some aspects, one or more capsid proteins of the AAV2 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions compared to the capsid proteins of native or wild-type AAV2 particles.

[0043] In certain embodiments, the rAAV9 particles comprise an AAV9 capsid. In certain embodiments, the rAAV9 particles are at least 60%, 65%, 70%, 75%, or more identical, 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 proteins of native or wild-type AAV9 particles, e.g., one or more capsid proteins (e.g., VP1, VP2, and / or VP3). In certain embodiments, the rAAV9 particles are at least 75% or more identical, e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to the capsid proteins VP1, VP2, and VP3 of native or wild-type AAV9 particles. In certain embodiments, the rAAV9 particles are variants of native or wild-type AAV9 particles. In some aspects, one or more capsid proteins of the AAV9 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions compared to the capsid proteins of native or wild-type AAV9 particles.

[0044] In certain embodiments, the rAAV particle contains one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, such as 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to the corresponding ITR of native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, or AAV-2i8, as long as the rAAV particle retains one or more desired ITR functions (e.g., DNA replication; integration of AAV DNA into the host cell genome, and / or, if desired, the ability to form a hairpin that enables packaging).

[0045] In certain embodiments, the rAAV2 particle contains one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, such as 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to the corresponding ITR of native or wild-type AAV2 particles, as long as the rAAV2 particle retains one or more desired ITR functions (e.g., DNA replication; integration of AAV DNA into the host cell genome; and / or, if desired, the ability to form a hairpin that enables packaging).

[0046] In certain embodiments, the rAAV9 particles include one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, such as 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical, to the corresponding ITRs of native or wild-type AAV2 particles, as long as they retain one or more desired ITR functions (e.g., DNA replication; integration of AAV DNA into the host cell genome; and / or, if desired, the ability to form hairpins that enable packaging).

[0047] The rAAV particles can include ITRs having any suitable number of "GAGC" repeats. In certain embodiments, the ITRs of the AAV2 particles include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more "GAGC" repeats. In certain embodiments, the rAAV2 particles include ITRs having 3 "GAGC" repeats. In certain embodiments, the rAAV2 particles include ITRs having fewer than 4 "GAGC" repeats. In certain embodiments, the rAAV2 particles include ITRs having more than 4 "GAGC" repeats. In certain embodiments, the ITRs of the rAAV2 particles include a Rep binding site where the fourth nucleotide of the first two "GAGC" repeats is C instead of T.

[0048] Exemplary suitable lengths of DNA that can be incorporated into an rAAV vector for packaging / encapsulation into rAAV particles can be about 5 kilobases (kb) or less. In specific embodiments, the length of the DNA is less than about 5 kb, less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, or less than about 2.5 kb.

[0049] An rAAV vector containing a nucleic acid sequence that directs the expression of RNAi or a polypeptide can be generated using appropriate recombinant techniques known in the art (see, e.g., Sambrook et al., 1989). Recombinant AAV vectors are typically packaged and propagated into transducible AAV particles using an AAV viral packaging system. Transducible AAV particles can bind to and enter mammalian cells and then deliver a nucleic acid cargo (e.g., a heterologous gene) to the nucleus of the cell. Thus, transducible intact rAAV particles are configured to transduce mammalian cells. rAAV particles configured to transduce mammalian cells are often replication-incompetent and require additional protein machinery for self-replication. Thus, rAAV particles configured to transduce mammalian cells are modified to bind to and enter mammalian cells and deliver nucleic acids to the cells, where the nucleic acids for delivery are often positioned between a pair of AAV ITRs within the rAAV genome.

[0050] Suitable host cells for generating transducible AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients of heterologous rAAV vectors. (For example, cells derived from the stable human cell line HEK293, readily available through the American Type Culture Collection under accession number ATCC CRL1573) can be used. In certain embodiments, modified human fetal kidney cell lines (e.g., HEK293) that are transformed with adenovirus type 5 DNA fragments and express the adenovirus E1a and E1b genes are used to generate recombinant AAV particles. The modified HEK293 cell line is readily transfected and provides a particularly convenient platform for generating rAAV particles. Methods for generating high-titer AAV particles that can be transduced into mammalian cells are known in the art. For example, AAV particles can be generated as described in Wright, 2008 and Wright, 2009.

[0051] In certain embodiments, the AAV helper function is introduced into the host cell by transfecting the host cell with an AAV helper construct either before or simultaneously with transfection of the AAV expression vector. Thus, AAV helper constructs may be used to complement the missing AAV functions necessary for productive AAV transduction and to provide at least transient expression of the AAV rep gene and / or cap gene. AAV helper constructs often lack AAV ITRs and cannot replicate or be packaged. These constructs can be in the form of plasmids, phages, transposons, cosmids, viruses, or virions. A number of AAV helper constructs are described, such as the commonly used plasmids pAAV / Ad and pIM29+45 that encode both Rep and Cap expression products. A number of other vectors encoding Rep and / or Cap expression products are known.

[0052] An "expression vector" is a special vector that contains a gene or nucleic acid sequence together with the necessary control regions required for expression in a host cell. The expression vector may at least contain an origin of replication for proliferation in the cell and optionally may contain additional elements such as heterologous nucleic acid sequences, expression regulatory elements (e.g., promoters, enhancers), introns, ITRs, and polyadenylation signals.

[0053] II. Therapeutic Agents In some embodiments, viral gene delivery methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding inhibitory RNAs, non-coding RNAs, and / or therapeutic proteins to cells in culture or within a host organism.

[0054] A. Inhibitory RNAs "RNA interference (RNAi)" is the process of sequence-specific post-transcriptional gene silencing initiated by siRNA. In RNAi, siRNA induces the degradation of target mRNA, and as a result, inhibits gene expression in a sequence-specific manner.

[0055] "Interfering RNA", "RNAi", "small interfering RNA", "short interfering RNA", or "siRNA" molecule, "short hairpin RNA" or "shRNA" molecule, or "miRNA" is an RNA duplex of nucleotides targeted to a nucleic acid sequence of interest. As used herein, the term "siRNA" is a generic term encompassing subsets 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 because 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 can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 base pairs in length. In some embodiments, the length of the duplex is 19 - 25 base pairs in length. In certain embodiments, the length of the duplex is 19 or 21 base pairs in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure can contain a loop portion located between the two sequences forming the duplex. The length of the loop can vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In certain embodiments, the loop is 18 nucleotides in length. The hairpin structure can also contain 3' and / or 5' overhang portions. In some embodiments, the overhang is a 3' and / or 5' overhang that is 0, 1, 2, 3, 4, or 5 nucleotides in length.

[0056] shRNAs are composed of a stem-loop structure designed to contain a 5' flanking region, siRNA region segment, loop region, 3' siRNA region, and 3' flanking region. Most RNAi expression strategies utilize short hairpin RNAs (shRNAs) driven by a strong polIII-based promoter. While many shRNAs have shown effective knockdown of target sequences both in vitro and in vivo, some shRNAs that have shown effective knockdown of target genes have also been found to be toxic in vivo.

[0057] miRNAs are small cellular RNAs (about 22 nt) processed from precursor stem-loop transcripts. Known miRNA stem-loops can be modified to contain an RNAi sequence specific to a gene of interest. Since miRNAs are endogenously expressed, miRNA molecules may be preferred over shRNA molecules. Thus, miRNA molecules have been shown to be less likely to induce the dsRNA-responsive interferon pathway, are processed more efficiently than shRNAs, and have an 80% higher silencing effect.

[0058] A recently discovered alternative approach is the use of artificial miRNAs (pri-miRNA scaffolds that shuttle siRNA sequences) as RNAi vectors. Artificial miRNAs are more natural, resemble endogenous RNAi substrates, and are more amenable to Pol-II transcription (e.g., enabling tissue-specific expression of RNAi) and polycistronic strategies (e.g., enabling delivery of multiple siRNA sequences). See U.S. Patent No. 10,093,927, which is incorporated by reference.

[0059] The transcription unit of 「shRNA」 consists of a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides. shRNA is exported from the nucleus by exportin 5 and, upon entering the cytoplasm, is processed by Dicer to generate functional siRNA. The 「miRNA」 stem-loop typically consists of a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides, expressed as part of a larger primary transcript (pri-miRNA), and is excised by the Drosha-DGCR8 complex to generate an intermediate known as pre-miRNA, which is then exported from the nucleus by exportin 5 and, upon entering the cytoplasm, is processed by Dicer to generate functional siRNA. 「Artificial miRNA」 or 「artificial miRNA shuttle vector」, as used interchangeably herein, refers to a primary miRNA transcript in which the region of the double-stranded stem-loop (at least about 9-20 nucleotides) excised via processing by Drosha and Dicer, while retaining the structural elements within the stem-loop required for efficient Drosha processing, has been replaced with an siRNA sequence targeting a target gene. The term 「artificial」 results from the fact that the flanking sequences (about 35 nucleotides upstream and about 40 nucleotides downstream) arise from restriction enzyme sites within the multiple cloning site of the siRNA. As used herein, the term 「miRNA」 encompasses both naturally occurring miRNA sequences and artificially generated miRNA shuttle vectors.

[0060] siRNA can be encoded by a nucleic acid sequence, which 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 the sequence of 6T.

[0061] In the design of RNAi, several factors need to be considered, such as the properties of siRNA, the persistence of the silencing effect, and the choice of delivery system. To produce the RNAi effect, the siRNA introduced into an organism typically contains exon sequences. Furthermore, since the RNAi process is homology-dependent, the sequences must be carefully selected to maximize gene specificity while minimizing the potential for cross-interference between homologous but non-gene-specific sequences. Preferably, the siRNA exhibits an identity greater than 80%, 85%, 90%, 95%, 98%, or even 100% between the sequence of the siRNA and the gene to be inhibited. Sequences with an identity of less than about 80% to the target gene have substantially lower effects. Therefore, the greater the homology between the siRNA and the gene to be inhibited, the lower the likelihood that the expression of unrelated genes will be affected.

[0062] Furthermore, the size of the siRNA is also an important consideration. In some embodiments, the present invention relates to siRNA molecules that contain at least about 19 to 25 nucleotides and are capable of modulating gene expression. For the present invention, the siRNA preferably has a length of less than 500, 200, 100, 50, or 25 nucleotides. More preferably, the siRNA is about 19 nucleotides to about 25 nucleotides in length.

[0063] An siRNA target generally refers to a polynucleotide that contains a region encoding a polypeptide, or a polynucleotide region that controls other processes important for replication, transcription, or translation, or the expression of a polypeptide, or a polynucleotide that contains both a region encoding a polypeptide and a region controlling expression that is functionally linked thereto. Any gene expressed in a cell can be targeted. Preferably, the target gene is one that is involved or associated with the progression of cell activities important for a disease, or is of particular interest as a research subject.

[0064] B. Non-coding RNA As demonstrated by cDNA cloning projects and genomic tiling arrays, more than 90% of the human genome is transcribed but does not encode proteins. These transcripts are called non-protein-coding RNAs (ncRNAs). A variety of ncRNA transcripts, such as ribosomal RNA, transfer RNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA), are essential for cellular functions. Similarly, numerous short ncRNAs, such as microRNA (miRNA), endogenous short interfering RNA (siRNA), PIWI-interacting RNA (piRNA), and small nucleolar RNA (snoRNA), are also known to play important regulatory roles in eukaryotic cells. Recent studies have demonstrated a group of long non-coding RNA (lncRNA) transcripts that exhibit cell-type specific expression and are localized to specific intracellular compartments. LncRNAs are also known to play important roles in cell development and differentiation, supporting the view that they have been selected during the process of evolution.

[0065] LncRNAs appear to have many different functions. In many cases, they either play a role in regulating protein activity or localization or function as an organizational framework for intracellular structures. In other cases, lncRNAs can be processed to generate multiple small RNAs or modulate how other RNAs are processed. The latest version of data generated by the public research consortium GenCode (version #27) lists nearly 16,000 lncRNAs within the human genome that generate nearly 28,000 transcripts; when including other databases, more than 40,000 lncRNAs are known.

[0066] Interestingly, lncRNAs can affect the expression of specific target proteins at specific genomic loci, modulate the activity of protein-binding partners, direct chromatin-modifying complexes to their sites of action, and undergo post-transcriptional processing to produce numerous small 5'-capped RNAs. Epigenetic pathways can also control the differential expression of lncRNAs.

[0067] Evidence is also accumulating that aberrantly expressed lncRNAs play important roles in both normal physiological processes and multiple disease states. lncRNAs are abnormally regulated in various diseases, such as ischemia, heart disease, Alzheimer's disease, psoriasis, and spinocerebellar ataxia type 8. This abnormal regulation has also been shown in various types of cancer, such as breast cancer, colon cancer, prostate cancer, hepatocellular carcinoma, and leukemia. Some lncRNAs, such as gadd74 and lncRNA-RoR5, modulate cell cycle regulators, such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and p53, and thus provide additional flexibility and robustness to cell cycle progression. Furthermore, some lncRNAs, such as centromeric satellite RNAs, which are essential for kinetochore formation and thus important for chromosome segregation during mitosis in humans and flies, are associated with the mitotic process. Another nuclear lncRNA, MA-lincl, functions in cis to control the exit from M phase by suppressing the expression of the neighboring gene Pura, which is a regulator of cell proliferation.

[0068] lncRNAs are generally defined as a group of transcripts that lack extended open reading frames (ORFs) and are more than 200 nucleotides in length (e.g., about 200 - about 1200 nt, about 2500 nt, or more). The term "non-coding RNA" (ncRNA) includes lncRNAs and also includes shorter transcripts, e.g., less than about 200 nt, such as about 30 - 200 nt.

[0069] Thus, in some embodiments, for example, delivery of an ncRNA to a particular brain structure of interest corrects abnormal RNA expression levels or modulates the levels of lncRNAs that cause disease. Thus, in some embodiments, the invention provides an rAAV in which the viral genome has been modified to encode a therapeutic non-coding RNA (ncRNA). In some embodiments, the ncRNA is a long non-coding RNA (lncRNA) having a length of about 200 nucleotides (nt) or more. In some embodiments, the therapeutic agent is an ncRNA having a length of about 25 nt or about 30 nt to about 200 nt. In some embodiments, the lncRNA has a length of about 200 nt to about 1,200 nt. In some embodiments, the lncRNA has a length of 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 nt.

[0070] C. CRISPR System Gene editing is a technology that enables modification of target genes in living cells. Recently, the utilization of the CRISPR bacterial immune system for on-demand gene editing has revolutionized the way scientists approach genome editing. The Cas9 protein of the CRISPR system, an RNA-guided DNA endonuclease, can be relatively easily modified to target new sites by changing its guide RNA sequence. This discovery has made sequence-specific gene editing functionally effective.

[0071] Generally, the term "CRISPR system" collectively refers to transcripts and other elements involved in the expression or regulation of the activity of CRISPR-associated ("Cas") genes, such as sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or the active portion of tracrRNA), tracr mate sequences (including "direct repeats" and partially processed direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts derived from the CRISPR locus.

[0072] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide RNA) that binds sequence-specifically to DNA and a Cas protein (e.g., Cas9) having nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a type I, type II, or type III CRISPR system from a particular organism, such as Streptococcus pyogenes, including, for example, an endogenous CRISPR system.

[0073] As described herein, the CRISPR system can induce double-strand breaks (DSBs) and subsequent disruption at a target site. In other embodiments, a Cas9 variant considered a "nickase" is used to introduce a single-strand nick at the target site. A pair of nickases each directed by a different pair of gRNAs targeting sequences such that 5' overhangs are introduced by simultaneous introduction of the nicks can be used, for example, to improve specificity. In other embodiments, Cas9 lacking catalytic activity is fused to a heterologous effector domain, such as a transcriptional repressor (e.g., KRAB) or activator, to affect gene expression. Alternatively, a CRISPR system comprising Cas9 lacking catalytic activity further includes a transcriptional repressor or activator fused to a ribosome-binding protein.

[0074] In some instances, a Cas nuclease and a gRNA (including a fusion of a crRNA specific for a target sequence and a tracrRNA that is immobilized) are introduced into a cell. Generally, the target site on the 5'-end side of the gRNA targets the Cas nuclease to a target site, such as a gene, using complementary base pairing. The target site can be selected based on a protospacer adjacent motif (PAM) sequence, for example, typically a position immediately 5' of NGG or NAG. In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, the CRISPR system is characterized by elements that facilitate the formation of the CRISPR complex at the site of the target sequence. Typically, a "target sequence" generally refers to a sequence that is designed such that the guide sequence has complementarity, and hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. Complete complementarity is not necessarily required, provided that there is sufficient complementarity to cause hybridization and facilitate the formation of the CRISPR complex.

[0075] The target sequence can include any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. The target sequence can be located in the nucleus or cytoplasm of a cell, such as within an organelle. 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" or "editing polynucleotide" or "editing sequence". In some instances, an exogenous template polynucleotide is also referred to as an editing template. In some instances, the recombination is homologous recombination.

[0076] Typically, for an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs, or more). A tracr sequence that comprises or can consist of all or a portion of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85 nucleotides, or more, of a wild-type tracr sequence, or about 20, 26, 32, 45, 48, 54, 63, 67, 85 nucleotides, or more, longer than a wild-type tracr sequence) can also form part of a CRISPR complex, e.g., by hybridization of at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has complementarity to a tracr mate sequence sufficient to hybridize and participate in formation of a CRISPR complex, e.g., having at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity to the full length of the tracr mate sequence when optimally aligned.

[0077] One or more vectors driving expression of one or more elements of a CRISPR system can be introduced into a cell such that expression of the elements of the CRISPR system directs formation of a CRISPR complex at one or more target sites. The components may be delivered to the cell as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence may each be operably linked to separate control elements in separate vectors. The Cas enzyme may be a target gene under the regulation of a controlled alternative splicing event disclosed herein as a chimeric target gene minigene or as the target gene of a chimeric minigene transactivator. The gRNA may be under the regulation of a constitutive promoter.

[0078] Alternatively, two or more of the elements expressed from the same or different control elements may be combined in a single vector, and one or more additional vectors may provide components of the CRISPR system that are not included in the first vector. The vector may include one or more insertion sites, for example, restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of the one or more vectors. When 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.

[0079] The vector may include a control element operably linked to an enzyme coding sequence that encodes a CRISPR enzyme, for example, 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. These enzymes are known; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0080] The CRISPR enzyme can be Cas9 (e.g., derived from Streptococcus pyogenes or Streptococcus pneumoniae (S. pneumoniae)). The CRISPR enzyme can direct cleavage of one or both strands at the position of the target sequence, e.g., within the target sequence and / or within the complementary strand of the target sequence. The vector can encode a CRISPR enzyme that is mutated compared to the corresponding wild-type enzyme such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, a substitution of aspartic acid to alanine (D10A) in the RuvC I catalytic domain of Cas9 derived from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence, e.g., two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination makes it possible to nick both strands and use it to induce NHEJ or HDR.

[0081] In some embodiments, the enzyme-encoding sequence encoding a CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. A eukaryotic cell can be of a particular organism, such as a mammal, such as, but not limited to, human, mouse, rat, rabbit, dog, or non-human primate, or derived therefrom. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species exhibit a particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage among organisms) is often correlated with the translation efficiency of messenger RNA (mRNA), which is thought to depend particularly on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs within a cell is generally a reflection of the codons most frequently used in peptide synthesis. Thus, based on codon optimization, genes can be tailored for optimal gene expression in a given organism.

[0082] Generally, a guide sequence is any polynucleotide sequence having complementarity with a target polynucleotide sequence sufficient to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more, or exceeds about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more when optimally aligned using a suitable alignment algorithm.

[0083] The optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which 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).

[0084] The CRISPR enzyme may be part of a fusion protein that includes one or more heterologous protein domains. The CRISPR enzyme fusion protein may include an additional protein sequence and, optionally, a linker sequence between the two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins such as blue fluorescent protein (BFP), but are not limited thereto. The CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that binds to a DNA molecule or another cellular molecule, such as, without limitation, maltose binding protein (MBP), S tag, Lex A DNA binding domain (DBD) fusion, GAL4A DNA binding domain fusion, and herpes simplex virus (HSV) BP16 protein fusion. Additional domains that can form part of a fusion protein containing the CRISPR enzyme are described in US 20110059502, which is incorporated herein by reference.

[0085] D. Therapeutic Proteins Some aspects relate to the expression of recombinant proteins and recombinant polypeptides. Such proteins can be neuroprotective proteins, such as anti-apoptosis proteins, antioxidant enzymes (e.g., those belonging to the superoxide dismutase (SOD) family), neurotrophic / neuroprotective factors, or anti-inflammatory proteins. Therapeutic proteins can be Birc1a (NAIP), Birc2 (c-IAP1 / HIAP-2), Birc3 (cIAP-2 / HIAP-1), Birc4 (XIAP), Birc5 (survivin), Birc6 (apollon), Birc7 (livin), Birc8 (TsIAP); members of the Bcl-2 family: Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bcl-2L10, BFL-1; c-Jun known as Jun interaction protein (JIP), JIP-1, JIP-2, JIP-3, JIP-4N-terminal kinase (JNK) endogenous inhibitor; SOD1, SOD2; catalase; peroxiredoxin 1, peroxiredoxin 2, glutathione preoxidase 1 (Gpx1), Gpx2, Gpx3, or Gpx4; NGF, BDNF, CNTF, GDNF, growth / differentiation factor 15 (GDF-15), erythropoietin, or vascular endothelial growth factor (VEGF); interleukin 10 (IL-10); glutathione S-transferase, annexin 1 (ANXA1), inhibitor of NF-κB (IκB); USH1, USH1G (alias SANS), CIB2 (calcium and integrin binding protein 2), ATOH-1, ACTG1, ATP2B2, CDH23 (cadherin 23), CLDN14, CLRN1, COCH, COL11A2, DFNA5, DFNB31, DFNB59, ESPN, EYA4, GJB2, GJB3, GJB6, KCNQ4, LHFPL5, MT-RNR1, MT-TS1, MYO1A, MYO6, MYO7A (myosin 7a), MYO15A, OTOF, PCDH15 (protocadherin 15), PDZD7, POU3F4, SLC26A4, STRC, TECTA, TMC1, TMC2, TMIE, TMPRSS3, TRIOBP, USH1C, VLGR1, WFS1, ACTG1, ADCY1, ATOFfl, ATP6V1B1, BDNF, BDP1, BSND, DATSPER2, CABP2, CD164, CDC14A, CDH23, CEACAM16, CHD7, CCDC50, CIB2, CLDN14, CLIC5, CLPP, CLRNl, COCH, COL2A1, COL4A3, COL4A4, COL4A5, COL9A1, COL9A2, COL11A1, COL11A2, CRYM, DCDC2, DFNA5, DFNB31, DFNB59, DIAPH1, EDN3, EDNRB, ELMOD3, EMOD3, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FAM65B, FOXI1, GIPC3, GJB2, GJB3, GJB6, GPR98, GRHL2, GPSM2, GRXCR1, GRXCR2, HARS2, HGF, HOMER2, HSD17B4, ILDR1, KARS, KCNE1, KCNJ10, KCNQ1, KCNQ4, KITLG, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MCM2, MET, MIR183, MIRN96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MY015A, MYOIA, MY03A, MY06, MY07A, NARS2, NDP, NF2, NT3, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, PNPT1, POLR1D, POLR1C, POU3F4, POU4F3, PRPS1, PTPRQ, RDX, S1PR2, SANS, SEMA3E, SERPINB6, SLC17A8, SLC22A4, SLC26A4, SLC26A5, SIX1, SIX5, S MAC / DIABLO, SNAI2, SOX10, STRC, SYNE4, TBC1D24, TCOF1, TECTA, TIMM8A, TJP2, TNC, TMC1, TMC2, TMIE, TMEM132E, TMPRSS3, TRPN, TRIOBP, TSPEAR, USH1C, USH1G, USH2A, USH2D, VLGRl, WFS1, WHRN, or XIAP.

[0086] Provided herein are methods and uses for administering to a mammal suspected of having or having a lysosomal storage disorder (LSD) in need of the methods described herein. In certain embodiments, the methods or uses described herein are used to treat, prevent, inhibit, reduce, decrease, or delay the number, severity, frequency, progression, or onset of one or more symptoms of LSD.

[0087] Non-limiting examples of LSDs include infantile lipofuscinosis or late-infantile neuronal ceroid lipofuscinosis (LINCL), Gaucher, juvenile Batten, Fabry, MLD, Sanfilippo A, late-infantile Batten, Hunter, Krabbe, Morquio, Pompe, Niemann-Pick C, Tay-Sachs, Hurler (MPS-I H), Sanfilippo B, Maroteaux-Lamy, Niemann-Pick A, cystinosis, Hurler-Scheie (MPS-I H / S), Sly syndrome (MPS VII), Scheie (MPS-I S), infantile Batten, GM1 gangliosidosis, mucolipidosis II / III, or Sandhoff disease.

[0088] LSDs are often caused by genetic abnormalities (e.g., mutations, deletions, insertions) in the gene encoding the tripeptidyl peptidase 1 (TPP1) enzyme, which lead to a deficiency in functional TPP1 enzyme activity. In humans, TPP1 is encoded by the CLN2 gene, which is sometimes called the TPP1 gene. For example, late-infantile neuronal ceroid lipofuscinosis (LINCL) is a pediatric neurodegenerative disorder most often caused by a deficiency in TPP1 activity due to mutations in CLN2. Development is normal until the age of 2-4 years, after which LINCL symptoms appear as motor and mental regression, episodic disorders, and visual impairment. Generally, death occurs within 10 years of birth. Most cases of LINCL are due to mutations in CLN2 that induce a deficiency in the soluble lysosomal enzyme tripeptidyl peptidase 1 (TPP1). TPP1, like other soluble lysosomal hydrolases, is synthesized as a mannose-6-phosphate enzyme precursor, and although most of this enzyme precursor is targeted to lysosomes, some is released from the cell via the secretory pathway. Therefore, cellular uptake by the same or neighboring cells, and subsequent lysosomal delivery and activation from the enzyme precursor to the active form, can occur.

[0089] In certain embodiments, provided herein is a method of treating a mammal having or suspected of having LSD by directly administering to the tissue or fluid of the central nervous system an AAV particle (referred to herein as an AAV-TPP1 particle) that directs the expression of a polypeptide having TPP1 activity.

[0090] When the present application refers to the function or activity of a "modified protein" or "modified polypeptide", one of ordinary skill in the art will understand that this includes, for example, a protein or polypeptide having additional advantages compared to an unmodified protein or polypeptide. Embodiments regarding "modified proteins" can be implemented with respect to "modified polypeptides" and vice versa is specifically contemplated.

[0091] A recombinant protein may have amino acid deletions and / or substitutions; thus, a protein having a deletion, a protein having a substitution, and a protein having both a deletion and a substitution are modified proteins. In some embodiments, these proteins may further include insertions or amino acid additions, such as, for example, a fusion protein or a protein having a linker. A "deletion-modified protein" may lack one or more residues of the native protein but may have the specificity and / or activity of the native protein. A "deletion-modified protein" may also have reduced immunogenicity or antigenicity. Examples of deletion-modified proteins are those having a deletion of amino acid residues from at least one antigenic region, i.e., a region of the protein determined to be antigenic in a particular organism, such as the organism to which the modified protein is administered.

[0092] A substitution variant or exchange variant typically contains the exchange of one amino acid for another at one or more sites within a protein and may be designed to modulate one or more properties of the polypeptide, specifically effector function and / or bioavailability. The substitution may be conservative, i.e., one amino acid may be exchanged for an amino acid of similar shape and charge, or it may not be. Conservative substitutions are known in the art and include, for example, changes from alanine to serine; from arginine to lysine; from asparagine to glutamine or histidine; from aspartic acid to glutamic acid; from cysteine to serine; from glutamine to asparagine; from glutamic acid to aspartic acid; from glycine to proline; from histidine to asparagine or glutamine; from isoleucine to leucine or valine; from leucine to valine or isoleucine; from lysine to arginine; from methionine to leucine or isoleucine; from phenylalanine to tyrosine, leucine, or methionine; from serine to threonine; from threonine to serine; from tryptophan to tyrosine; from tyrosine to tryptophan or phenylalanine; and from valine to isoleucine or leucine.

[0093] In addition to deletions or substitutions, the modified protein may also have an insertion of residues, typically including the addition of at least one residue, within the polypeptide. This can include the insertion of a targeting peptide or targeting polypeptide, or the insertion of only a single residue. Terminal additions, called fusion proteins, are described below.

[0094] The term "biologically functionally equivalent" is well understood in the art and is further defined in more detail herein. Thus, provided that the biological activity of the protein is maintained, sequences are included in which about 70% to about 80%, or about 81% to about 90%, or even about 91% to about 99% of the amino acids are identical or functionally equivalent to the amino acids of the control polypeptide. Recombinant proteins can be biologically functionally equivalent to their native counterparts in certain aspects.

[0095] Amino acid sequences and nucleic acid sequences, even if they contain additional residues, such as additional N-terminal or C-terminal amino acids, or 5' or 3' sequences, in terms of protein expression, as long as the sequences meet the above criteria, including the maintenance of biological protein activity, it will also be understood that they can essentially be those described in one of the sequences disclosed herein. The addition of terminal sequences specifically applies to nucleic acid sequences that may contain various non-coding sequences adjacent to either the 5' or 3' portion of the coding region, for example, or various internal sequences known to exist within a gene, i.e., nucleic acid sequences that may contain introns.

[0096] As used herein, a protein or peptide generally refers to a protein from the translation of a gene, up to a full-length sequence greater than about 200 amino acids; a polypeptide greater than about 100 amino acids; and / or a peptide of about 3 to about 100 amino acids, although not limited thereto. For convenience, the terms "protein", "polypeptide", and "peptide" are used interchangeably herein.

[0097] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimetic known in the art. In certain embodiments, the residues of a protein or peptide are contiguous without any non-amino acids interrupting the sequence of amino acid residues. In other embodiments, the sequence may include one or more non-amino acid moieties. In specific embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0098] Accordingly, the term "protein or peptide" encompasses an amino acid sequence that includes at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or rare amino acid.

[0099] Certain embodiments of the invention relate to fusion proteins. These molecules can have a therapeutic protein linked to a heterologous domain at the N-terminus or C-terminus. For example, the fusion may utilize a leader sequence from another species to enable recombinant expression of the protein in a heterologous host. Another useful fusion includes the addition of a protein affinity tag, preferably cleavable, such as a serum albumin affinity tag or 6 histidine residues, or an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST).

[0100] Methods for generating fusion proteins are well known to those of skill in the art. Such proteins may be made, for example, by de novo synthesis of a complete fusion protein or by attachment of a DNA sequence encoding the heterologous domain and subsequent expression of the intact fusion protein.

[0101] The production of a fusion protein that recovers the functional activity of a parent protein can be facilitated by connecting genes with a cross-linking DNA segment that encodes a peptide linker spliced between tandemly connected polypeptides. The linker is of sufficient length to allow proper folding of the resulting fusion protein.

[0102] Expression of the transgene may be directed by the transgene's native promoter (i.e., the promoter naturally found with the transgene coding sequence), or expression of the transgene may be directed by a heterologous promoter (e.g., CMV promoter, espin promoter, PCDH15 promoter, PTPRQ promoter, and TMHS (LHFPL5) promoter). For example, any of the transgenes described herein can be used with its native promoter. Alternatively, any of the transgenes described herein can be used with a heterologous promoter. As used herein, a heterologous promoter refers to a promoter that does not naturally direct the expression of its sequence (i.e., is not naturally found with that sequence). Representative heterologous promoters that can be used to direct the expression of any of the transgenes shown herein include, for example, CMV promoter, CBA promoter, CASI promoter, P promoter, and EF-1 promoter, α9 nicotinic receptor promoter, prestin promoter, Gfil promoter, and Vglut3 promoter. Further, a promoter that naturally directs the expression of one of the above-described transgenes (e.g., KCNQ4 promoter, Myo7a promoter, Myo6 promoter, or Atohl promoter) may be used as a heterologous promoter to direct the expression of the transgene. In other embodiments, the promoter is an espin promoter, PCDH15 promoter, PTPRQ promoter, and TMHS (LHFPL5) promoter.

[0103] III. Methods of Treatment and Administration Viral vectors may, in some situations, be administered directly to a patient (in vivo), or used to treat cells in vitro or ex vivo and then 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 agent that can be engineered by insertion or incorporation of nucleic acid. A vector, e.g., a viral vector, can be used to introduce (introduce / transfer) a nucleic acid sequence into a cell such that, when the nucleic acid sequence therein is transcribed and encodes a protein, it is then translated by the cell.

[0104] Any suitable cell or mammal can be administered or treated by the methods or uses described herein. Typically, a mammal in need of the methods described herein is presumed to have or express an abnormal or aberrant protein associated with a disease state. Alternatively, a mammalian recipient may be in a state receptive to 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 in situ expression of the administered genetic material. Thus, the phrase "state receptive to gene replacement therapy" encompasses conditions such as genetic diseases (i.e., disease states resulting from one or more gene defects), acquired pathologies (i.e., pathological states not resulting from congenital defects), cancer, and prophylactic procedures (i.e., prevention of a disease or unwanted medical condition). Thus, as used herein, the term "therapeutic agent" refers to any agent or material that has a beneficial effect on a mammalian recipient. Thus, "therapeutic agents" include both therapeutic and prophylactic molecules having nucleic acid or protein components.

[0105] Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domesticated animals (e.g., dogs and cats), livestock (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In certain embodiments, the mammal is a human. In certain embodiments, the mammal is a non-rodent mammal (e.g., human, pig, goat, sheep, horse, dog, etc.). In certain embodiments, the non-rodent mammal is a human. The mammal can be of any age or any developmental stage (e.g., adult, teenager, child, infant, or mammalian fetus in utero). The mammal can be male or female. In certain embodiments, the mammal can be an animal disease model, e.g., an animal model having or expressing an abnormal protein associated with a disease state, or an animal model having insufficient protein expression that causes a disease state.

[0106] Mammals (subjects) treated by the methods or compositions described herein include adults (18 years of age or older) and children (less than 18 years of age). Adults include the elderly. A representative adult is 50 years of age or older. The age of children ranges from 1 - 2 years, or 2 - 4 years, 4 - 6 years, 6 - 18 years, 8 - 10 years, 10 - 12 years, 12 - 15 years, and 15 - 18 years. Children include infants. Infants typically range from 1 - 12 months of age.

[0107] In certain embodiments, the method comprises administering a plurality of viral particles to a mammal as described herein, wherein the severity, frequency, progression, or onset time of one or more symptoms of a disease state, such as a neurodegenerative disease, is decreased, reduced, prevented, inhibited, or delayed. In certain embodiments, the method comprises administering a plurality of viral particles to a mammal for treating a deleterious symptom of a disease state, such as a neurodegenerative disease. In certain embodiments, the method comprises administering a plurality of viral particles to a mammal for stabilizing a disease state, such as a neurodegenerative disease, for delaying or preventing its worsening or progression, or for reversing its deleterious symptoms.

[0108] In certain embodiments, the method comprises administering a plurality of viral particles to the central nervous system of a mammal or a portion thereof as described herein, wherein the severity, frequency, progression, or onset time of one or more symptoms of a disease state, such as a neurodegenerative disease, is decreased, reduced, prevented, inhibited, or delayed by at least about 5 to about 10 days, about 10 to about 25 days, about 25 to about 50 days, or about 50 to about 100 days.

[0109] In certain embodiments, the symptom or deleterious effect is an early, intermediate, or late symptom; a behavioral, personality, or language symptom; a swallowing, motor, seizure, tremor, or fidgeting symptom; ataxia; and / or a cognitive symptom, such as memory, the ability to organize.

[0110] IV. Pharmaceutical Compositions As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" mean biologically acceptable compositions, formulations, liquids, or solids, or mixtures thereof, that are suitable for one or more routes of administration, in vivo delivery, or contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material can be administered to a subject without causing substantial undesirable biological effects. Such compositions, "pharmaceutically acceptable" and "physiologically acceptable" formulations and compositions can be sterile. Such pharmaceutical formulations and compositions can be used, for example, in the administration of viral particles to a subject.

[0111] Such formulations and compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, media for dispersions and suspensions, coatings, isotonic agents, and absorption promoters or retardants that are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions can include suspending and thickening agents. Supplementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents, and antifungal agents) can also be incorporated into the formulations and compositions.

[0112] Pharmaceutical compositions typically contain pharmaceutically acceptable excipients. Such excipients include any pharmaceutical agent that, by itself, does not induce the production of antibodies harmful to the individual receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts, such as mineral acid salts, e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids, e.g., acetate, propionate, malonate, benzoate, etc., may be included therein. Further, auxiliary substances, such as surfactants, wetting or emulsifying agents, pH buffering substances, etc., may be present in such media.

[0113] The pharmaceutical composition can be formulated to be compatible with a particular route of administration or delivery, as described herein or as known to those skilled in the art. Accordingly, the pharmaceutical composition includes carriers, diluents, or excipients suitable for administration or delivery by various routes.

[0114] Pharmaceutical forms suitable for injection or infusion of virus particles can include sterile aqueous solutions or dispersions that are suitable for the immediate preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the final form should be a sterile liquid and stable under the conditions of manufacture, use, and storage. The liquid carrier or medium can be, for example, a solvent or liquid dispersion medium including water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the formation of liposomes, in the case of dispersions, by maintaining the required particle size, or by the use of surfactants. Isotonic agents, such as sugars, buffers, or salts (such as sodium chloride), can be included. Prolonged absorption of the injectable composition can be brought about by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0115] 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, an aqueous saline solution, such as phosphate buffered saline (PBS), artificial CSF, a surfactant, a non-volatile oil, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), glycerin, or other synthetic solvents; antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, etc.; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetic acid, citric acid, or phosphoric acid, and agents for adjusting osmotic pressure, such as sodium chloride or dextrose.

[0116] Pharmaceutical formulations, compositions, and delivery systems suitable for the compositions, methods, and uses of the present invention are known in the art (see, for example, 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), R.L. Juliano, ed., Oxford, N.Y., pp. 253-315).

[0117] Viral particles and their compositions can be formulated into dosage unit forms for ease of administration and uniformity of dosage. As used herein, a dosage unit form refers to a physically discrete unit suitable as a unit dosage for an individual to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect, together with the necessary pharmaceutical carrier. The dosage unit form depends on the number of viral particles considered necessary to produce the desired effect. The required amount may be formulated as a single dose or as multiple dosage units. The dosage is adjusted to an appropriate viral particle concentration and may optionally be combined with an anti-inflammatory agent and packaged for use.

[0118] In one aspect, the pharmaceutical composition contains a therapeutically effective amount, i.e., an amount sufficient to reduce or alleviate the symptoms or adverse effects of the disease state, or an amount sufficient to confer the desired benefit, of genetic material.

[0119] As used herein, "unit dosage form" refers to a physically discrete unit suitable as a unit dosage for a subject to be treated, and each unit contains a predetermined amount calculated to produce the desired effect (e.g., a prophylactic or therapeutic effect) when administered in one or more doses, optionally together with a pharmaceutical carrier (excipient, diluent, medium, or filler). The unit dosage form may be, for example, in ampoules and vials, which may contain a liquid composition or a composition in a freeze-dried or lyophilized state; for example, a sterile liquid carrier may be added before in vivo administration or delivery. Individual unit dosage forms may be included in a multi-dose kit or container. Thus, for example, viral particles and their pharmaceutical compositions can be packaged into single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0120] Formulations containing virus particles typically contain an effective amount readily determined by one of ordinary skill in the art. The virus particles may typically range from about 1% to about 95% (w / w) of the composition, or may be even higher if appropriate. The amount administered depends on factors such as the age, weight, and physical condition of the mammalian or human subject being considered for treatment. An effective dosage can be established by one of ordinary skill in the art through routine testing to establish a dose-response curve.

[0121] V. Definitions The terms "polynucleotide," "nucleic acid," and "transgene" are used interchangeably herein to refer to all forms of nucleic acids, oligonucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and their polymers. Polynucleotides include genomic DNA, cDNA, and antisense DNA, as well as spliced or unspliced mRNA, rRNA, tRNA, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin-type (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA, or antisense RNA). Polynucleotides can include naturally occurring polynucleotides, synthetic polynucleotides, and polynucleotides that have been intentionally modified or altered (e.g., variant nucleic acids). A polynucleotide can be single-stranded, double-stranded, or triple-stranded, linear or circular, and of any suitable length. In the description of a polynucleotide, the sequence or structure of a particular polynucleotide can be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0122] Nucleic acids encoding polypeptides often contain an open reading frame encoding the polypeptide. Unless otherwise indicated, a particular nucleic acid sequence includes degenerate codon substitutions.

[0123] The nucleic acid may comprise one or more expression regulatory elements or expression control elements operably linked to an open reading frame, wherein the one or more control elements are configured to direct transcription and translation of a polypeptide encoded by the open reading frame in mammalian cells. Non-limiting examples of expression regulatory / control elements include transcription initiation sequences (e.g., promoters, enhancers, TATA box, etc.), translation initiation sequences, mRNA stability sequences, polyA sequences, secretion sequences, etc. The expression regulatory / control elements can be obtained from the genome of any suitable organism.

[0124] "Promoter" refers to a nucleotide sequence, generally upstream (5') of the coding sequence, that directs and / or regulates the expression of a coding sequence by providing recognition for RNA polymerase and other factors necessary for proper transcription. A pol II promoter includes a minimal promoter that is a short DNA sequence composed of a TATA box and optionally other sequences that functions to specify the transcription start site to which control elements are added for regulation of expression. A type 1 pol III promoter includes three cis-acting sequence elements: (a) a 5' sequence element (A block); (b) an intermediate sequence element (I block); (c) a 3' sequence element (C block), downstream of the transcription start site. A type 2 pol III promoter includes two essential cis-acting sequence elements: (a) an A box (5' sequence element); and (b) a B box (3' sequence element), downstream of the transcription start site. A type 3 pol III promoter includes several cis-acting promoter elements, such as a conventional TATA box, a proximal sequence element (PSE), and a distal sequence element (DSE), upstream of the transcription start site.

[0125] An "enhancer" is a DNA sequence capable of stimulating transcriptional activity and can be an endogenous element of a promoter or a heterologous element that enhances the level or tissue specificity of expression. It can function in either direction (5'->3' or 3'->5') and can function when located either upstream or downstream of a promoter.

[0126] A promoter and / or enhancer may be entirely derived from a native gene, or may be composed of different elements derived from different elements found in nature, or may even be composed of synthetic DNA segments. A promoter or enhancer may contain DNA sequences involved in the binding of protein factors that modulate / regulate the effectiveness of transcription initiation in response to stimuli, physiological, or developmental conditions.

[0127] Non-limiting examples of promoters include the SV40 early promoter, the mouse mammary tumor virus LTR promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, e.g., the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, pol II promoters, pol III promoters, synthetic promoters, hybrid promoters, and the like. Further, sequences derived from non-viral genes, such as the mouse metallothionein gene, are also useful herein. Exemplary constitutive promoters include the promoters of the following genes that encode certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, the actin promoter, U6, and other constitutive promoters known to those of skill in the art. Further, many viral promoters function constitutively in eukaryotic cells. These include, inter alia, the early and late promoters of SV40; the long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses; and the thymidine kinase promoter of herpes simplex virus. Thus, any of the above-described constitutive promoters can be used to regulate the transcription of a heterologous gene insert.

[0128] As used herein, the term "transgene" is used for convenience to refer to a nucleic acid sequence / polynucleotide that is intended or has been introduced into a cell or organism. A transgene includes any nucleic acid, e.g., a gene encoding an inhibitory RNA or a polypeptide or protein, and is generally heterologous to the naturally occurring AAV genomic sequence.

[0129] The term "transducing" refers to the introduction of a nucleic acid sequence into a cell or host organism by a vector (e.g., a viral particle). Thus, the introduction of a transgene into a cell by a viral particle can be referred to as "transduction" of the cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. When the introduced transgene is integrated into the nucleic acid (genomic DNA) of the recipient cell or recipient organism, it is stably maintained in that cell or organism and can further be inherited or passed on to progeny cells or progeny organisms of the recipient cell or recipient organism. Finally, the introduced transgene may exist extrachromosomally or only transiently in the recipient cell or host organism. Thus, a "transduced cell" is a cell into which a transgene has been introduced by transduction. Thus, a "transduced" cell is a cell or its progeny into which a transgene has been introduced. Transduced cells can be propagated and the transgene can be transcribed and the encoded inhibitory RNA or protein can be expressed. In the case of the use and methods of gene therapy, transduced cells can be present in a mammal.

[0130] Transgenes under the regulation of inducible promoters are expressed only in the presence of an inducer or more so in the presence of an inducer (e.g., transcription under the regulation of the metallothionein promoter is greatly increased in the presence of certain metal ions). Inducible promoters contain response elements (REs) that stimulate transcription when an inducer factor binds. For example, there are REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. Promoters containing specific REs can be selected to obtain an inducible response, and in some cases, the RE itself can be attached to a different promoter, thereby conferring inducibility to the recombinant gene. Thus, by selecting an appropriate promoter (constitutive or inducible; strong or weak), it is possible to regulate both the presence and expression level of a polypeptide in genetically modified cells. When a gene encoding a polypeptide is under the regulation of an inducible promoter, in situ delivery of the polypeptide is induced by exposing the in situ genetically modified cells to conditions that allow transcription of the polypeptide, for example, by intraperitoneal injection of a specific inducer of the inducible promoter that regulates transcription of the drug. For example, in situ expression of a polypeptide encoded by a gene under the regulation of the metallothionein promoter by genetically modified cells is enhanced by contacting the genetically modified cells in situ with a solution containing the appropriate (i.e., inducing) metal ion.

[0131] A nucleic acid / transgene is "functionally linked" when placed in a functional relationship with another nucleic acid sequence. A nucleic acid / transgene encoding RNAi or a polypeptide, or a nucleic acid that directs the expression of a polypeptide, may contain an inducible or tissue-specific promoter to regulate the transcription of the encoded polypeptide. A nucleic acid functionally linked to an expression regulatory element can also be called an expression cassette.

[0132] In certain embodiments, cell type-specific or inducible promoters, enhancers, etc. are utilized in the methods and uses described herein. Non-limiting examples of cell type-specific promoters include those isolated from genes derived from TMC1, TMC2, espin, PCDH15, PTPRQ, TMHS (LHFPL5), MYO1A. Non-limiting examples of inducible promoters include DNA response elements for ecdysone, tetracycline, hypoxia, and IFN.

[0133] In certain embodiments, the expression regulatory element comprises a CMV enhancer. In certain embodiments, the expression regulatory element comprises a β-actin promoter. In certain embodiments, the expression regulatory element comprises a chicken β-actin promoter. In certain embodiments, the expression regulatory element comprises a CMV enhancer and a chicken β-actin promoter.

[0134] As used herein, the terms "modify" or "variant" and their grammatical variations mean that a nucleic acid, polypeptide, or a subsequence thereof deviates from a reference sequence. Thus, modified and variant sequences may have substantially the same, more, or less expression, activity, or function as the reference sequence, but retain at least a 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, such as a missense mutation or a nonsense mutation.

[0135] A variant of a "nucleic acid" or "polynucleotide" refers to a modified sequence that has been genetically altered compared to the wild type. The sequence can be genetically modified without changing the encoded protein sequence. Alternatively, the sequence can be genetically modified to encode a variant protein. A variant of a nucleic acid or polynucleotide can also refer to a combinatorial sequence that is codon-modified to encode a protein that still retains at least partial sequence identity to a reference sequence, e.g., a wild-type protein sequence, and is codon-modified to encode a variant protein. For example, some codons of such a nucleic acid variant have changed without changing the amino acid of the protein encoded thereby, and some codons of such a nucleic acid variant have changed such that the amino acid of the protein encoded thereby has changed.

[0136] The terms "protein" and "polypeptide" are used interchangeably herein. The "polypeptides" encoded by the "nucleic acids" or "polynucleotides" or "transgenes" disclosed herein include partial or full-length native sequences, naturally occurring wild-type and functional polymorphic proteins, their functional partial sequences (fragments), and their sequence variants, as long as such polypeptides retain some function or activity. Thus, in the methods and uses of the present invention, such polypeptides encoded by nucleic acid sequences need not be identical to the defective endogenous protein, or the endogenous protein whose activity, function, or expression is insufficient, absent, or non-existent, in the mammal being treated.

[0137] Non-limiting examples of modifications include substitutions of one or more nucleotides or amino acids (e.g., about 1 to about 3, about 3 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 500, about 500 to about 750, about 750 to about 1000, or more nucleotides or residues).

[0138] Examples of amino acid modifications are conservative amino acid substitutions or deletions. In certain embodiments, the modified sequence or variant sequence retains at least a portion of the function or activity of the unmodified sequence (e.g., wild-type sequence).

[0139] Another example of an amino acid modification is the introduction of a targeting peptide to the capsid protein of a viral particle. Recombinant viral vectors have been identified with peptides that target the central nervous system, e.g., distinct brain regions.

[0140] Such modified recombinant viruses can preferentially bind to one type of tissue (e.g., CNS tissue) over another type of tissue (e.g., liver tissue). In certain embodiments, a recombinant virus carrying a modified capsid protein can "target" cerebrovascular epithelial tissue by binding at higher levels than a comparable unmodified capsid protein. For example, a recombinant virus with a modified capsid protein can bind to cerebrovascular epithelial tissue at levels 50% to 100% higher than an unmodified recombinant virus.

[0141] A "nucleic acid fragment" is a portion of a given nucleic acid molecule. In most organisms, deoxyribonucleic acid (DNA) is the genetic material, and ribonucleic acid (RNA) is involved in the transfer of information contained within DNA to proteins. Fragments and variants of the disclosed nucleotide sequences, as well as the proteins or partial-length proteins encoded thereby, are also encompassed by the present invention. "Fragment" or "portion" means all or less than all of a nucleotide sequence encoding a polypeptide or protein, or an amino acid sequence of a polypeptide or protein. In certain embodiments, the fragment or portion is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the wild-type activity or function).

[0142] A "variant" of a molecule is a sequence that is substantially similar to the sequence of the native molecule. In the case of a nucleotide sequence, variants include sequences that encode the same amino acid sequence of the native protein due to the degeneracy of the genetic code. Naturally occurring allelic variants, for example, can be identified by the use of molecular biology techniques such as polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences include nucleotide sequences obtained synthetically, such as nucleotide sequences generated by using site-directed mutagenesis, which encode the native protein, and also include those that encode polypeptides having amino acid substitutions. Generally, nucleotide sequence variants of the present invention have at least 40%, 50%, 60% - 70%, for example, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% - 79%, generally at least 80%, for example, 81% - 84%, at least 85%, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% - 98% sequence identity to the native (endogenous) nucleotide sequence. In certain embodiments, the variant is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the wild-type activity or function).

[0143] A "conservative variant" of a particular nucleic acid sequence refers to a nucleic acid sequence that encodes the same or essentially the same amino acid sequence. Due to the degeneracy of the genetic code, a number of functionally identical nucleic acids encode a given polypeptide. For example, the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at all positions where arginine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded protein. Such nucleic acid variations are "silent variations," a type of "conservatively modified variation." All nucleic acid sequences described herein that encode a polypeptide, unless otherwise noted, also describe all possible silent variations. One of ordinary skill in the art will recognize that each codon within a nucleic acid (except for ATG, which is typically the only codon for methionine) can be modified by standard techniques to provide a functionally identical molecule. Thus, each "silent variation" of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0144] The term "substantial identity" of polynucleotide sequences means that a polynucleotide compared to a reference sequence using one of the alignment programs described using standard parameters has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity. One of ordinary skill in the art will recognize that these values can be appropriately adjusted to determine the corresponding identity of the proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes typically means at least 70%, at least 80%, 90%, or even at least 95% sequence identity.

[0145] With respect to a polypeptide, the term "substantial identity" indicates that the polypeptide contains a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98%, or 99% sequence identity to a reference sequence in a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunologically reactive with an antibody raised against the second polypeptide. Thus, for example, if two peptides differ only by conservative substitutions, the polypeptide is identical to the second polypeptide.

[0146] "Force sensation" means a response to a mechanical stimulus. Examples of the conversion of a mechanical stimulus into a neuronal signal are touch, hearing, and the sense of balance. Force-sensory input is converted into a response to a mechanical stimulus through a process called "mechanotransduction".

[0147] "Disease" means a condition or disorder that damages cells, tissues, or organs or interferes with their normal function. Examples of diseases include, for example, genetic diseases characterized by a loss of function of a protein that functions in force sensation and is expressed in the inner ear of a subject. In another aspect, the disease is Usher syndrome (e.g., USH1) or presbycusis. In one aspect, the disease is a hearing disorder associated with a gene defect, e.g., a defect in TMC1, TMC2, MYO7A, USH1C, CDH23, PCDH15, SANS, CIB2, USH2A, VLGR1, WHKN, CLRN1, PDZD7, USH1C (e.g., harmonin a, b, or c).

[0148] As used herein, the terms "treating" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures aimed at preventing, inhibiting, reducing, or decreasing an undesirable physiological change or disorder, e.g., the onset, progression, or worsening of a disorder. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, which may or may not be detectable, diminishment of the degree of a disease, stabilization of a disease state (i.e., absence of worsening or progression), delay or slowing of disease progression, remission or amelioration of a disease state, and recovery (partial or complete). "Treatment" can also mean an extension of survival as compared to expected survival if not receiving treatment. A person in need of treatment includes not only a person already having the condition or disorder, but also a person having a predisposition thereto (e.g., determined by a genetic assay).

[0149] As used herein, "essentially free of" with respect to a specified component means that the specified component is not intentionally formulated in the composition and / or is present only as an impurity or in trace amounts. Thus, the total amount of the specified component due to unintentional contamination of the composition is far below 0.05%, preferably below 0.01%. Most preferably, the composition is one in which the amount of the specified component cannot be detected by standard analytical methods.

[0150] As used herein, "a" or "an" can mean one or more. As used in the claims, when used in conjunction with the word "comprising", the words "a" or "an" can mean one or more.

[0151] The use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer only to alternatives or unless the alternatives are expressly stated to be mutually exclusive, although the disclosure supports definitions that refer only to alternatives and to "and / or". As used herein, "another" can mean at least a second or more.

[0152] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error of the device used to determine the value, the inherent variation of the method, the variation that exists between subjects being studied, or a value within 10% of the recited value.

[0153] VI. Kit The present invention provides a kit comprising a packaging material and one or more components contained therein. The kit typically includes a label or packaging insert containing instructions for the components or for the in vitro, in vivo, or ex vivo use of the components. The kit can contain a collection of such components, such as nucleic acids, recombinant vectors, and / or viral particles.

[0154] A kit refers to a physical structure that houses one or more components of the kit. The packaging material can be made of materials commonly used for such purposes that can maintain the components aseptically (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0155] The label or insert may include identification information of one or more components therein, dosage, clinical pharmacology of the active ingredient, for example, mechanism of action, pharmacokinetics, and pharmacodynamics. The label or insert may include information identifying the manufacturer, lot number, place and date of manufacture, and expiration date. The label or insert may include information identifying the manufacturer information, lot number, place and date of the manufacturer. The label or insert may include information regarding the diseases for which the components of the kit can be used. The label or insert may include instructions for the physician or the subject regarding the use of one or more of the components of the kit in a method, use, or treatment protocol or treatment plan. The instructions may include the amount, frequency, or duration of administration, and instructions for implementing any of the methods, uses, treatment protocols, or prevention or treatment plans described herein.

[0156] The label or insert may include information regarding any benefits that the component may provide, for example, preventive or therapeutic benefits. The label or insert may include information regarding possible adverse side effects, complications, or reactions, for example, warnings to the subject or physician regarding situations in which the use of a particular composition is not appropriate. Adverse side effects or complications may occur when the subject is receiving, is scheduled to receive, or is currently receiving one or more other drug therapies that may be incompatible with the composition, or when the subject is receiving, is scheduled to receive, or is currently receiving another treatment protocol or treatment plan that may be incompatible with the composition, and thus the instructions may include information regarding such incompatibilities.

[0157] Labels or inserts include "printed matter", such as paper or cardboard, that are separate from, attached to, or contained within components of the kit, or packaging materials (e.g., boxes), or attached to ampoules, tubes, or vials that contain components of the kit. Labels or inserts may further include computer-readable media, such as printed labels with barcodes, disks, optical disks, such as CDs or DVD-ROM / RAM, DVDs, MP3s, or electrical storage media, such as RAM and ROM, or hybrids thereof, such as magnetic / optical storage media, flash memory, hybrids, and memory type cards.

Example

[0158] VII. Example The following examples are included to illustrate preferred embodiments of the present invention. The techniques disclosed in the following examples represent techniques that have been found by the inventors to function well in the practice of the present invention and are thus considered to constitute preferred modes for their practice. However, one of ordinary skill in the art will understand that, in light of the present disclosure, many changes can be made to the specific embodiments disclosed and still obtain similar or analogous results without departing from the spirit and scope of the present invention.

[0159] Example 1 - Delivery of AAV Vector to the Blood-Brain Barrier in Mice Brain endothelial cells (BECs) are a major component of the blood-brain barrier, which is a major barrier to drug delivery to the central nervous system. Adeno-associated virus (AAV) targeted to BECs not only directly treats diseases that affect these cells but also has the potential to deliver secreted proteins across the blood-brain barrier to the central nervous system (CNS). AAV variants that efficiently transduce mouse BECs after systemic administration are known, and these variants can be used to treat mouse models of diseases that affect the CNS, such as lysosomal storage disorders. However, systemic AAV administration requires high doses and exposes the periphery to AAV virions and transgenes, which can result in toxicity or an immune response.

[0160] Here, the inventors tested whether similar transduction could be achieved after intracerebroventricular injection (ICV) at significantly lower doses compared to systemic injection. For this purpose, in C57BL / 6J mice, FVB / NJ mice, and Tpp1 knockout mice, which are a lysosomal storage disease model of CLN2 deficiency, a diverse library of peptide-modified AAVs (PM-AAVs) was screened by two selections after ICV injection (Figure 1A). Amplicon sequencing of the AAV capsid sequences revealed the most enriched variants in the isolated BECs (Figures 1B - 1D). Two variants were AAV9 capsids with targeting peptide sequences SNVVRAT (SEQ ID NO:25) and RTNDTKL (SEQ ID NO:26). After sequencing, two PM-AAV variants expressing EGFP were assayed after injection into the ICV or retroorbitally in C57BL / 6J and FVB / NJ mice at a total dose of 2E10 vector genomes. In mice injected unilaterally by ICV, bilateral EGFP expression was observed in all CD31-positive endothelial cells of the brain and spinal cord (Figures 2A - 2F). EGFP expression was also evident, particularly in some neuronal and glial cells close to the injection site and in the liver (Figures 3A - 3D). Surprisingly, mice injected retroorbitally instead of by ICV at the same dose showed similar or improved transduction into BECs, with minimal EGFP expression observed in other brain cell types (Figures 4A - 4I). Transduction into the liver was comparable between the two administration routes (Figure 3D vs. Figure 4I). Collectively, these results suggest that the PM-AAV variants delivered by ICV escaped from the CNS to the periphery rather than remaining in the CNS as originally intended and transduced BECs from the lumen of cerebral blood vessels. Systemic administration of AAV is likely to be a preferred route of administration for gene therapy targeting cerebral blood vessels. However, the PM-AAVs identified here showed highly efficient transduction into BECs at a relatively low total dose, with minimal expression of EGFP in other brain cell types. These PM-AAV variants will be valuable tools for studying BEC function and gene therapy for CNS diseases.

[0161] Example 2 - Delivery of AAV Vectors to the Blood - Brain Barrier in Primates A peptide - modified AAV library was generated by inserting random peptides into AAV1, AAV2, and AAV9 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. AAV enrichment studies specific to the cerebral microvasculature were performed. In each round of enrichment, an NGS amplicon sequencing library was generated, and sequencing data was used to track the enrichment of each capsid construct. Table 1 provides peptide candidates for AAV1. Table 2 provides peptide candidates for AAV2. Table 3 provides peptide candidates for AAV9.

[0162] (Table 1) AAV1 - targeting Peptides TIFF2025516715000001.tif114160

[0163] (Table 2) AAV2 - targeting Peptides TIFF2025516715000002.tif50160

[0164] (Table 3) AAV9 - targeting Peptides TIFF2025516715000003.tif149160

[0165] VGLMVSR (SEQ ID NO:7) was ranked as a top hit by enrichment fold over input in the AAV1 DNA results, but was detected at much lower levels (0.06% of all UMIs) than the other AAV1 candidate TGVLINR (0.2%; SEQ ID NO:8) and TGALVIR (0.1%; SEQ ID NO:9). VGLMVSR (SEQ ID NO:7) was also not detected at high levels in the RNA dataset, in contrast to which the robustly expressed AAV1 sequences were only the two of TGALVIR (SEQ ID NO:9) and TGVLINR (SEQ ID NO:8). From these features, TGVLINR (SEQ ID NO:8) and TGALVIR (SEQ ID NO:9) were the most attractive AAV1 candidates for proceeding to immediate validation.

[0166] The selected capsid candidates are advanced to fluorescence-based validation.

[0167] All of the methods disclosed herein and claimed in the claims can be made and carried out without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods and processes or the order of the steps described herein without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that the same or similar results may be achieved by substituting chemically and physiologically related certain agents for the agents described herein. All such similar substitutes and modifications apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

Claims

**Claim 1** A modified AAV capsid protein comprising a targeting peptide that targets a viral vector containing a modified adeno-associated virus (AAV) capsid protein to brain endothelial cells, wherein the targeting peptide is 3 to 10 amino acids in length. **Claim 2** The modified AAV capsid protein according to claim 1, having any one of the sequences of SEQ ID NO: 26, 27, 25, and 28-42. **Claim 3** The modified AAV capsid protein according to claim 1, which is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein. **Claim 4** The modified AAV capsid protein according to claim 1, wherein the modified AAV capsid protein is derived from the AAV1 capsid protein (see SEQ ID NO: 1), and the targeting peptide is inserted after residue 590 of the AAV1 capsid protein. **Claim 5** The modified AAV capsid protein according to claim 4, wherein a linker sequence is adjacent to the targeting peptide, and the linker sequences on each side of the targeting peptide are 2 or 3 amino acids in length. **Claim 6** The modified AAV capsid protein according to claim 5, wherein the linker sequence on the N-terminal side of the targeting peptide is SSA, and the linker sequence on the C-terminal side of the targeting peptide is AS. **Claim 7** The modified AAV capsid protein according to claim 6, wherein the modified AAV1 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:

4. **Claim 8** The modified AAV capsid protein according to claim 7, wherein the targeting peptide is any one of SEQ ID NO: 8, 9, 7, and 10-12. **Claim 9** The modified AAV capsid protein according to claim 1, wherein the modified AAV capsid protein is derived from the AAV2 capsid protein (see SEQ ID NO: 2), and the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. **Claim 10** The modified AAV capsid protein according to claim 9, wherein a linker sequence is adjacent to the targeting peptide, and the linker sequences on each side of the targeting peptide are 2 or 3 amino acids in length. **Claim 11** The modified AAV capsid protein according to claim 10, wherein the linker sequence on the N-terminal side of the targeting peptide is AAA and the linker sequence on the C-terminal side of the targeting peptide is AA.

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

5.

13. The modified AAV capsid protein according to claim 12, wherein the targeting peptide is any one of SEQ ID NOs: 13 to 15.

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

15. The modified AAV capsid protein according to claim 14, wherein a linker sequence is adjacent to the targeting peptide and the linker sequences on each side of the targeting peptide are 2 or 3 amino acids in length.

16. The modified AAV capsid protein according to claim 15, wherein the linker sequence on the N-terminal side of the targeting peptide is AAA and the linker sequence on the C-terminal side of the targeting peptide is AS.

17. The modified AAV capsid protein according to claim 16, wherein the modified AAV9 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:

6.

18. The modified AAV capsid protein according to claim 17, wherein the targeting peptide is any one of SEQ ID NOs: 16 to 26.

19. The modified AAV capsid protein according to claim 1, wherein the target peptide contains a sequence of a maximum of 10 amino acids in length having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 26 internally.

20. The modified AAV capsid protein according to claim 19, wherein the targeting peptide is 7 amino acids in length.

21. A nucleic acid comprising a sequence encoding the modified capsid protein according to any one of claims 1 to 20.

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

23. A viral vector comprising a nucleic acid encoding the modified capsid protein according to any one of claims 1 to 20.

24. The viral vector according to claim 23, further comprising a nucleic acid sequence encoding a nucleic acid of interest.

25. The viral vector according to claim 24, wherein the nucleic acid of interest is a therapeutic agent.

26. The viral vector according to claim 25, wherein the therapeutic agent is a protein or an RNAi molecule.

27. A cell comprising the viral vector according to any one of claims 23 to 26.

28. The cell according to claim 27, which is a mammalian cell.

29. The cell according to claim 27, which is a human cell.

30. The cell according to claim 27, which is in vitro.

31. The cell according to claim 27, which is in vivo.

32. A pharmaceutical composition comprising the virus according to claim 22 and a pharmaceutically acceptable carrier.

33. A method for delivering a drug to the cerebral microvasculature of a subject, comprising the step of administering the virus according to claim 22 to the subject.

34. The method according to claim 33, wherein the drug is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, a therapeutic protein, or a CRISPR system.

35. The method according to any one of claims 33 to 34, wherein the administration is administration to the central nervous system.

36. The method according to claim 35, wherein the administration is administration into the cisterna magna, the intracerebroventricular cavity, the mantle, the ventricle, the subarachnoid space, and / or the intrathecal space.

37. The method according to any one of claims 33 to 34, wherein the administration is systemic administration.

38. The method according to any one of claims 33 to 37, wherein a plurality of virus particles are administered.

39. The virus is about 1 x 10 per kilogram 6 ~Approx. 1×10 18 40. The method of claim 38, wherein the dose is administered in units of vector genome (vg / kg).

40. The virus is administered at a dose of about 1×10 7 to 1×10 17 about 1×10 8 to 1×10 16 about 1×10 9 to 1×10 15 about 1×10 10 to 1×10 14 about 1×10 10 to 1×10 13 about 1×10 10 to 1×10 13 about 1×10 10 to 1×10 11 about 1×10 11 to 1×10 12 about 1×10 12 to ×10 13 or about 1×10 13 to 1×10 14 vg / kg, according to the method of claim 38.

41. The method according to any one of claims 33 to 40, wherein the subject is a human.

42. A method for treating a disease in a mammal, comprising the step of administering the virus according to claim 22 to the mammal.

43. The method according to claim 42, wherein the disease is a disease affecting the central nervous system.

44. The method according to claim 43, wherein the disease is lysosomal storage disease, Glut1 deficiency, or multiple sclerosis.

45. The method according to claim 42, wherein the mammal is a human.

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