Compositions and methods

Abstract

The present invention relates to adeno-associated virus (AAV) capsid proteins that have been modified to insert amino acid sequences and / or methods of targeting microglia or brain macrophages using the AAV capsid proteins of the invention.

Description

[Technical Field]

[0001] This invention involves the insertion of amino acid sequences. ru kai The present invention relates to a modified adeno-associated virus (AAV) capsid protein, and / or a method for targeting microglia or brain macrophages using the AAV capsid protein of the present invention. [Background technology]

[0002] Microglia are tissue-resident macrophages of the central nervous system (CNS) and play a crucial role in CNS immune defense, development, and homeostasis (Schafer and Stevens, 2015; Li and Barres, 2018; Salter and Stevens, 2017; Wolf et al., 2017; Prinz et al., 2019). These highly dynamic cells are constantly responding to their environment (Gosselin et al., 2017). Genetic studies have also strongly associated microglial dysfunction with neurodegeneration, neuroinflammation, and neuropsychiatric and neurodevelopmental states (Guerreiro et al., 2013; Jonsson et al., 2013; Tansey, Cameron, and Hill, 2018; Gjoneska et al., 2015; Young et al., 2019). Functionally, microglia appear to lose their capabilities with age, particularly in the context of neurodegenerative and neuroinflammatory states. Specific examples of this include chronic demyelinating diseases such as multiple sclerosis (MS), where the ability of microglia to aggregate at injury sites and induce phagocytosis of necrotic tissue fragments decreases with age (Kotter et al., 2006; Natrajan et al., 2015; Ruckh et al., 2012). Furthermore, human studies have shown that microglia may be a harmful element in MS, and that the presence of large numbers of activated microglial cells is an early phenomenon preceding demyelination and lesion formation, correlated with chronic impairment.

[0003] In relation to neurodegeneration, specific changes in microglial function have been linked to Parkinson's disease and Alzheimer's disease, and pro-inflammatory microglia have been shown to be associated with amyloid-beta (Aβ) plaques. Furthermore, decreased phagocytic activity of microglia is a characteristic feature of amyotrophic lateral sclerosis (Wolf et al., 2017). In relation to neuropsychiatric disorders, changes in microglial density and morphology in distinct brain regions have been shown in patients with autism spectrum disorder, while abnormalities in microglial activation accompanied by neuroinflammatory changes have been observed in patients with schizophrenia (Leza et al., 2015).

[0004] Microglia are an attractive therapeutic target for the central nervous system (CNS) primarily due to their abundance and self-renewal capabilities. Currently, only limited therapeutic interventions exist, such as nonspecific therapies like CSF1R inhibitors, which have been used in animal models of Alzheimer's disease (Spangenberg et al., 2019). However, recent studies have revealed that microglia exist in various functional states in both animal models and humans (Young et al., 2019; Olah et al., 2018; Keren-Shaul et al., 2017; Hammond et al., 2019; Masuda et al., 2019; Mrdjen et al., 2018; Mathys et al., 2017), making a more attractive proposal to selectively and precisely edit the functional genes of relevant cell subpopulations.

[0005] The development of the PHP.B plasmid has enabled highly efficient CNS infection via systemic circulation (Deverman et al., 2016). More recently, PHP.eB has been used for gene editing of stem cells in the CNS (Segel et al., 2019). While PHP.eB has been shown to be effective in transduction of neurons, it has not been proven that in vivo infection of microglia with PHP.eB is achievable (Deverman et al., 2016; Kumar et al., 2020).

[0006] Therefore, there is a need for improved compositions and methods for targeting microglia in vivo. This invention addresses this need by providing novel AAV capsid proteins and AAV particles that can invade microglia and resident brain macrophages, including recruited monocyte-derived CNS-infiltrating macrophages. [Overview of the Initiative]

[0007] This invention is based on the creation of a novel AAV capsid protein that can be introduced into AAV virus particles. AAVs possessing this modified capsid protein can cross the blood-brain barrier and efficiently target cells throughout the CNS, including microglia and brain macrophages, including recruited monocyte-derived CNS-infiltrating macrophages.

[0008] These novel AAV capsid proteins insert amino acid sequences into the binding arms of the AAV capsid. ru kai A modification has been made. The novel AAV capsid protein of the present invention can be introduced into viral particles that are particularly effective in targeting microglial cells. Thus, AAVs containing the novel AAV capsid protein of the present invention are highly advantageous compared to the aforementioned AAVs because they exhibit efficient transduction in cells throughout the CNS.

[0009] Therefore, the present invention provides the following: Insert the amino acid sequence having the following characteristics. ru kai Modified adeno-associated virus (AAV) capsid protein: (a) The amino acid sequence of SEQ ID NO: 1, or a variant thereof having one amino acid substitution; (b) The amino acid sequence of Sequence ID No. 2, or a variant thereof having one amino acid substitution; (c) The amino acid sequence of SEQ ID NO: 3, or a variant thereof having one amino acid substitution; (d) The amino acid sequence of SEQ ID NO: 4, or a variant thereof having one amino acid substitution; (e) The amino acid sequence of Sequence ID No. 5, or a variant thereof having one amino acid substitution; (f) The amino acid sequence of SEQ ID NO: 6, or a variant thereof having one amino acid substitution; (g) The amino acid sequence of Sequence ID No. 7, or a variant thereof having one amino acid substitution; (h) The amino acid sequence of Sequence ID No. 8, or a variant thereof having one amino acid substitution; or, (i) Amino acid sequence of Sequence ID No. 9.

[0010] Furthermore, the present invention provides the following: A nucleic acid encoding the AAV capsid protein according to the present invention; recombinant DNA containing the nucleic acid according to the present invention; a host cell containing the nucleic acid or recombinant DNA according to the present invention; a viral particle containing the AAV capsid protein according to the present invention; a host cell producing the viral particle according to the present invention; a non-human transgenic animal containing the viral particle according to the present invention; or a pharmaceutical composition comprising the AAV capsid protein, nucleic acid, or viral particle according to the present invention and one or more pharmaceutically acceptable excipients.

[0011] Furthermore, the present invention provides the following: A method for targeting microglia or brain macrophages using the AAV capsid protein according to the present invention, comprising introducing a recombinant AAV vector into a mammal that encodes a target gene, a gene editing construct, an antibody or antigen-binding fragment, or a gene silencing construct, encapsulated in the capsid protein according to the present invention. [Brief explanation of the drawing]

[0012] [Figure 1]Figure 1: Explanation of vector design for creating an infectious random library. (A) Schematic diagram of PHP.eB AAV containing a random library insertion. (B) Transfer vector used to create a novel virus library. The PHP.eB capsid sequence is split at amino acid position 588, and the PHP.eB binding arm is replaced by a 21-base pair random library capable of generating 1.9448e9 (×109) different viruses. This plasmid further encodes a GFP fluorescent tag co-expressed with the capsid gene to identify infected cells. (C) The rep gene and cap gene required for AAV production are provided in an additional plasmid. The cap gene used for library screening contains three silencing sites to prevent the expression of the VP1, VP2, and VP3 capsid proteins of AAV9. [Figure 2] Figure 2: Infection of microglia (Iba-1) is not shown even when CNS tissue is infected with the PHP.eB capsid and the GFP transgene (GFP), as confirmed by the absence of double-positive (yellow) cells. [Figure 3] Figure 3: Infection of CNS tissue with a random library. (A) Infection of microglia (Iba-1) is shown by the GFP transgene (GFP), as confirmed by the presence of double-positive (yellow) cells. (B) FACS plot of microglia infection (PE / 561) and GFP transgene (GFP / 488). Double-positive cells (PE+ / GFP+) highlight microglia / intracerebral macrophage cells that were able to be infected with the above virus. [Figure 4-1]Figure 4: Characterization of four novel binding arms regarding AAV entry into microglia. (A) Representative FACS plot showing the infection rate (%) of microglia using one of the novel viruses containing the random library insertion HGTAASH. (B) Representative FACS plot showing the infection rate (%) of microglia using one of the novel viruses containing the random library insertion ALAVPFR. (C) Representative FACS plot showing the infection rate (%) of microglia using one of the novel viruses containing the random library insertion ALAVPFK. (D) Representative FACS plot showing the infection rate (%) of microglia using one of the novel viruses containing the random library insertion YAFGGEG. [Figure 4-2] Same as above. [Figure 5] Figure 5: Microglia isolated and cultured from the brain after in vivo infection (scale: 50 μM). [Figure 6] Figure 6: Comparison of brain entry of the novel capsid (HGTAASH) against AAV9 and PHP.eB. C57 / BL6 mice were infected with AAV9 encoding a GFP reporter (Addgene), PHP.eB + GFP reporter (Addgene), and a novel virus (HGTAASH) having a binding arm HGTAASH encoding a mCherry reporter. After infection with 5 × 1011 viral particles, the mice were bred for 4 weeks to allow protein expression. (a) Injection of AAV-9 showed no uptake throughout the brain parenchyma. (b) Injection of PHP.eB showed uptake in 42% of brain cells other than microglia, but not in microglia. (c) The HGTAASH capsid invaded 17% of brain cells other than microglia and 75% of microglia. (d–f) Immunohistochemical staining of brain sections of (d) AAV9 + GFP reporter, (e) PHP.eB + GFP reporter, and (f) HGTAASH vector + mCherry reporter (M3 microglia vector). Iba-1 microglia (white) and related reporter gene; GFP (green) or mCherry (red). Scale bar: 10 μM. [Figure 7]Figure 7: Expression of gain-of-function transgenes in microglia. C57 / BL6 mice were injected with a diphtheria toxin (DTA) transgene under the control of the human CD11b promoter. (a) Flow cytometry showed a significant decrease in microglia in samples after DTA transgene expression compared to control mice injected with the mCherry reporter. (b) Immunohistochemistry confirmed a decrease in the number of microglia in tissue sections. (c) High-magnification images of microglia in samples injected with the mCherry reporter showed quiescent microglia in contrast to apoptotic microglia in samples injected with the DTA-containing virus. Scale bars: (b) 50 μM; (c) 10 μM. [Figure 8] Figure 8: Induction of functional downregulation in microglia. B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP) mice were injected with viruses encoding mCherry under the control of the human Cd11b promoter, and control shRNA or shRNA designed against GFP, both under the control of the U6 promoter. (a) Flow cytometry confirmed a 75% microglial infection rate, as previously reported. Furthermore, (b) in mice injected with GFP-targeting shRNA, 58% of post-infection microglia showed decreased GFP levels. (c) Immunohistochemistry confirmed decreased GFP expression in mCherry-positive microglia. Iba-1 microglia (red) and associated reporter gene; GFP (green) or mCherry (yellow). Scale bar: 10 μM. [Figure 9]Figure 9: CRISPR gene editing of microglia. B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP) mice were injected with a virus encoding a guide RNA for GFP driven by the U6 promoter. (a) Flow cytometry showed a microglial infection rate of 75%, as previously reported. Furthermore, (b) in mice injected with GFP-targeting guide RNA, 17% of post-infection microglia showed a decrease in GFP levels. (c) Immunohistochemistry confirmed a decrease in GFP expression in mCherry-positive microglia. Iba-1 microglia (red) and associated reporter gene; GFP (green) or mCherry (yellow). Scale bar: 10 μM. [Figure 10] Figure 10: In vitro infection of human microglia. Microglia were isolated from 8-12 week old human embryos. Microglia-specific capsid virus encoding the mCherry reporter under the control of the human Cd11b promoter (HGTAASH) was added to the culture medium at a concentration of 1 × 10⁹, and the infection rate was compared with that of PHP.eB. Immunocytochemistry shows mCherry expression in cultured human microglia compared to infection with PHP.eB. Scale bar: 10 μM. [Figure 11] Figure 11: Random capsid sequences can be inserted into AAV2 and AAV6. The novel insertion sequence HGTAASH was inserted into the capsid sequences of other AAV serotypes, and the potential for microglial infection by these modified capsids was tested. Microglial infection-compatible, characterized binding arms were cloned into AAV2 and AAV6. Microglia were isolated from C57 / BL6 mice as previously reported. AAVs with modified capsid sequences were added to culture medium at a concentration of 1 × 10⁹. Immunocytochemistry showed that modified AAV2 and modified AAV6 were associated with mCherry transgene expression in cultured mouse microglia, in contrast to PHP.eB, which is known to readily infect the CNS but not microglia. Scale bar: 10 μM.

[0013] A brief explanation of the table. Table 1: Table of PCR primers for DNA amplification. Table 2: Table of sequences designed for integration into plasmids. Table 3: Table showing five additional novel binding arms. Sequences that enable infection by resident brain microglia after insertion into the capsid protein were identified by Sanger sequencing, and nucleotide sequence translation was performed in computer. * This shows peptide sequences that occur frequently. Table 4: A table of additional novel binding arm sequences capable of infecting microglia, identified using Illumina sequencing.

[0014] A brief explanation of arrays Sequence ID 1 shows the amino acid sequence of insertion sequence 1. Sequence ID 2 shows the amino acid sequence of insertion sequence 2. Sequence ID 3 shows the amino acid sequence of insertion sequence 3. Sequence ID 4 shows the amino acid sequence of insertion sequence 4. Sequence ID 5 shows the amino acid sequence of insertion sequence 5. Sequence ID 6 shows the amino acid sequence of insertion sequence 6. Sequence ID 7 shows the amino acid sequence of insertion sequence 7. Sequence ID 8 shows the amino acid sequence of insertion sequence 8. Sequence ID 9 shows the amino acid sequence of insertion sequence 9. Sequence ID 10 represents the wild-type sequence in PHP.eB. Sequence ID 11 shows the amino acid sequence of novel capsid protein 1, including insertion sequence 1. Sequence ID 12 shows the amino acid sequence of novel capsid protein 2, including insert sequence 2. Sequence ID 13 shows the amino acid sequence of novel capsid protein 3, including insert sequence 3. Sequence ID 14 shows the amino acid sequence of novel capsid protein 4, including insert sequence 4. Sequence ID 15 shows the amino acid sequence of novel capsid protein 5, including insert sequence 5. Sequence ID 16 shows the amino acid sequence of novel capsid protein 6, including insert sequence 6. Sequence ID 17 shows the amino acid sequence of novel capsid protein 7, including the inserted sequence 7. Sequence ID 18 shows the amino acid sequence of novel capsid protein 8, including the inserted sequence 8. Sequence ID 19 shows the amino acid sequence of novel capsid protein 9, including the inserted sequence 9. Sequence ID 20 shows the DNA sequence of novel capsid protein 1, including insertion sequence 1. Sequence ID 21 shows the DNA sequence of novel capsid protein 2, including insertion sequence 2. Sequence ID 22 shows the DNA sequence of novel capsid protein 3, including insertion sequence 3. Sequence ID 23 shows the DNA sequence of novel capsid protein 4, including insertion sequence 4. Sequence ID 24 shows the DNA sequence of novel capsid protein 5, including insertion sequence 5. Sequence ID 25 shows the DNA sequence of novel capsid protein 6, including insertion sequence 6. Sequence ID 26 shows the DNA sequence of novel capsid protein 7, including the inserted sequence 7. Sequence ID 27 shows the DNA sequence of novel capsid protein 8, including the inserted sequence 8. Sequence ID 28 shows the DNA sequence of novel capsid protein 9, including the inserted sequence 9. Sequence ID 29 shows the DNA sequence of insertion sequence 1. Sequence ID 30 shows the DNA sequence of insertion sequence 2. Sequence ID 31 shows the DNA sequence of insertion sequence 3. Sequence ID 32 shows the DNA sequence of insertion sequence 4. Sequence ID 33 shows the DNA sequence of insertion sequence 5. Sequence ID 34 shows the DNA sequence of insertion sequence 6. Sequence ID 35 shows the DNA sequence of insertion sequence 7. Sequence ID 36 shows the DNA sequence of insertion sequence 8. Sequence ID 37 shows the DNA sequence of insertion sequence 9. Sequence IDs 38 and 39 show primer sequences that hybridize to the PHP.eB replication center. Sequence IDs 40 and 41 show primer sequences that hybridize to the 3' capsid protein of PHP.eB. Sequence IDs 42 and 43 show primer sequences that hybridize to T2A-GFP. Sequence IDs 44 and 45 show primer sequences that hybridize to the PHP.eB backbone. Sequence IDs 46 and 47 show primer sequences that hybridize to the PHP.eB binding arm. Sequence ID 48 shows the DNA sequence of a random library fragment. Sequence ID 49 shows the DNA sequence of the silenced PHP.eB capsid protein. Sequence ID 50 shows the DNA sequence of the split capsid protein, including the DNA sequence of insertion sequence 9. Sequence ID 51 shows the DNA sequence of the split capsid protein, including the DNA sequence of insertion sequence 2. Sequence ID 52 shows the DNA sequence of the split capsid protein, including the DNA sequence of insertion sequence 3. Sequence ID 53 shows the DNA sequence of the split capsid protein, including the DNA sequence of insertion sequence 1. Sequence IDs 54-207 show the amino acid sequences of the additional, newly inserted sequences. Sequence IDs 208-361 show the DNA sequences of the additional, novel insertion sequences. Sequence ID 362 shows the amino acid sequence of AAV6. Sequence ID 363 shows the amino acid sequence of AAV2. Sequence ID 364 shows the amino acid sequence of AAV6 containing HGTAASH. Sequence ID 365 shows the amino acid sequence of AAV2 containing HGTAASH. [Modes for carrying out the invention]

[0015] As used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural form unless otherwise specifically indicated by the context. For example, "a polynucleotide" includes "multiple capsid proteins," "a polynucleotide" includes "multiple polynucleotides," "a nucleic acid" includes "multiple nucleic acids," "a promoter" includes "multiple promoters," and "a viral particle" includes two or more such viral particles.

[0016] All publications, patents, and patent applications cited herein, whether mentioned above or below, are incorporated herein by reference in their entirety.

[0017] The present invention relates to an improved AAV capsid protein for improving cell transduction. AAVs comprising the capsid protein of the present invention can be used to deliver targeted gene expression throughout the CNS and to silence the expression and / or activity of genes involved in neurodegeneration.

[0018] AAV Capsid Protein Compared to the wild-type AAV capsid protein, the AAV capsid protein of the present invention is modified. Such AAV capsid proteins are functional capsid proteins that have the ability to form AAV virus particles that can infect and / or transduce cells. Functional capsid proteins are capable of encapsulating genetic material, entering cells, and transducing the genetic material into cells. In particular, AAV virus particles containing the AAV capsid protein of the present invention are capable of infecting and / or transducing cells throughout the CNS, such as microglia. Those skilled in the art can easily determine whether a modified AAV capsid protein is a functional capsid protein using methods known in the art. Exemplary methods for investigating the functionality of AAV capsid proteins are described herein.

[0019] The AAV capsid protein of the present invention can be obtained from any adeno-associated virus (AAV) or its derivatives. As is well known to those skilled in the art, naturally occurring AAV viruses can be classified based on various biological systems.

[0020] The AAV genome typically includes packaging genes such as rep genes and / or cap genes that encode the packaging function of the AAV virus particle. The rep genes encode one or more of the Rep78 protein, Rep68 protein, Rep52 protein, and Rep40 protein, or their variants. The cap genes encode one or more capsid proteins, such as VP1, VP2, and VP3, or their variants, including the gene encoding the modified capsid protein of the present invention. These proteins constitute the capsid of the AAV virus particle. The AAV capsid protein of the present invention may have a modification to any of VP1, VP2, and / or VP3. In a preferred embodiment, the AAV capsid protein of the present invention has a modification to VP1.

[0021] Typically, AAV viruses are given names related to their serotype. A serotype corresponds to a variant of AAV that has a specific reactivity that can be used to distinguish it from other variants based on the expression profile of its capsid surface antigen. Typically, viruses with a particular AAV serotype do not efficiently cross-react with neutralizing antibodies specific to any other AAV serotype. Examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as recombinant serotypes such as Rec2 and Rec3, which have recently been identified from primate brains. The AAV capsid protein of the present invention can be obtained from any AAV serotype. The AAV capsid protein of the present invention may also be a chimeric capsid protein. For example, the AAV capsid protein of the present invention may contain amino acid sequences derived from at least one, at least two, or at least three different AAV serotypes. The numbering of amino acid residues may differ between AAV serotypes.

[0022] Reviews of AAV serotypes can be found in Choi et al. (Curr Gene Ther., 2005; Vol. 5 (No. 3); pp. 299-310) and Wu et al. (Molecular Therapy., 2006; Vol. 14 (No. 3), pp. 316-327). The sequences of the AAV genome, or components of the AAV genome such as the cap gene, for use in this invention can be obtained from the accession numbers of the following AAV whole genome sequences: adeno-associated virus 1 NC_002077, AF063497; adeno-associated virus 2 NC_001401; adeno-associated virus 3 NC_001729; adeno-associated virus 3B NC_001863; adeno-associated virus 4 NC_001829; adeno-associated virus 5 Y18065, AF085716; adeno-associated virus 6 NC_001862; avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; avian AAV strain DA-1 NC_006263, AY629583; bovine AAV NC_005889, AY388617.

[0023] In one embodiment of the present invention, the AAV capsid protein of the present invention is obtained from any CNS-targeting AAV. In related embodiments of the present invention, the AAV capsid protein of the present invention is obtained from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 5 (AAV5), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype rh10 (AAVrh10), retrograde AAV (AAVRetrograde), or PHP.eB. The AAV capsid protein of the present invention is most preferably obtained from PHP.eB, a derivative of AAV9.

[0024] In one embodiment of the present invention, a modified AAV capsid protein having an insertion sequence in the binding arm is a wild-type PHP.eB capsid protein having the sequence of SEQ ID NO: 10. The modified AAV capsid protein of the present invention also includes variants of SEQ ID NO: 10 that have a different sequence from SEQ ID NO: 10 but retain the ability to form viral particles that can infect and / or transduce microglia or brain macrophages. Such sequences have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 10.

[0025] The variant sequence identity percentage is preferably measured over the full length of the corresponding portion of SEQ ID NO: 10 aligned with the variant sequence, or over a section of SEQ ID NO: 10 consisting of at least 400, 500, 600, or 700 consecutive amino acids.

[0026] In a preferred embodiment of the present invention, the AAV capsid protein inserts the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 9. ru kaiModifications have been made. The insertion sequences of the present invention also include variants of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 9 that differ in sequence from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 9, but retain the ability to form a functional AAV capsid protein when inserted into an AAV capsid, and that form viral particles that infect and / or transduce microglia or brain macrophages. Such insertion sequences may have at least one amino acid substitution, at least two amino acid substitutions, or at least three amino acid substitutions. Exemplary variant sequences include SEQ ID NOs: 147-150, 162-164, and 176-181. Such insertion sequences may further include additional amino acids that extend beyond the sequences provided herein. That is, the insertion sequences of the present invention may be at least 7 amino acid lengths, at least 8 amino acid lengths, at least 9 amino acid lengths, or at least 10 amino acid lengths.

[0027] As described in the examples, the inventors have identified additional insertion sequences that, when inserted into the AAV capsid, retain the ability to form a functional AAV capsid protein and form viral particles that infect and / or transduce microglia or brain macrophages. These additional sequences are shown in Table 4 below. That is, in another aspect of the present invention, any of the sequences shown in Table 4 may be used to infect and / or transduce microglia or brain macrophages. For example, the AAV capsid protein of the present invention may have any of the amino acid sequences of the insertion sequences (SEQ ID NOs. 54-207) shown in Table 4 inserted into it. ru kai Changes may occur.

[0028] Insertion site The AAV capsid protein of the present invention inserts the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 9, or any variant thereof as described above. ru kaiModifications have been made. The amino acid sequences of SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, or 9, or their variants as described above, can be inserted into any desired section of the AAV capsid protein. In one exemplary embodiment, the variant has one of the amino acid sequences of SEQ ID NOs. 147-150, 162-164, and 176-181 and is inserted into any desired section of the AAV capsid protein. In the context of infection and / or transduction of microglia or brain macrophages, any of the insert sequences (SEQ ID NOs. 54-207) shown in Table 4 can be inserted into any desired section of the AAV capsid protein.

[0029] In a preferred embodiment, the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 9, or any variant thereof as described above, is inserted between amino acids 588 and 589 of SEQ ID NO: 10. For example, the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 9, or any one of SEQ ID NOs: 147-150, 162-164, and 176-181, is inserted between amino acids 588 and 589 of SEQ ID NO: 10. In the context of infection and / or transduction of microglia or brain macrophages, any of the insertion sequences shown in Table 4 (SEQ ID NOs: 54-207) is inserted between amino acids 588 and 589 of SEQ ID NO: 10.

[0030] To avoid any misunderstanding, it should be stated that the indication that the insertion site is amino acid X means that the binding peptide is inserted between amino acid X and amino acid X+l (i.e., the binding peptide is inserted after the indicated amino acid).

[0031] In another embodiment, the AAV capsid protein of the present invention inserts the amino acid sequence of SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, or 9, or a variant thereof as described above, at an equivalent position of a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO. 10. ru kai Modifications are made. For example, any one of the amino acid sequences of SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, or 9, or SEQ ID NOs. 147-150, 162-164, and 176-181, is inserted at an equivalent position of a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 10. In the context of infection and / or transduction of microglia or brain macrophages, any of the amino acid sequences of the insertion sequences shown in Table 4 (SEQ ID NOs. 54-207) may be inserted at an equivalent position of a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 10.

[0032] The amino acid position numbering described herein corresponds to the amino acid positions of the parent AAV sequence. The amino acid positions of the parent PHP.eB sequence obtained from AAV9 are described herein. Equivalent positions are those equivalent to positions 588 and 589 of SEQ ID NO: 10 (i.e., equivalent to positions 588 and 589 of the modified wild-type PHP.eB). Those skilled in the art can easily identify equivalent positions using methods known in the art. In particular, equivalent positions can be identified by sequence alignment. For example, in a given sequence, equivalent positions can be identified by aligning the sequence with the sequence of SEQ ID NO: 10 and identifying the positions equivalent to positions 588 and 589 of SEQ ID NO: 10. In one exemplary embodiment, equivalent positions are positions 487-488 of AAV6 (SEQ ID NO: 362). In one exemplary embodiment, equivalent positions are positions 586-587 of AAV2 (SEQ ID NO: 363). Based on the above, modified AAV6 capsid proteins and modified AAV2 capsid proteins containing the novel insertion sequence HGTAASH are shown as SEQ ID NOs. 364 and 365, respectively.

[0033] In one exemplary embodiment, the AAV capsid protein of the present invention has the amino acid sequence of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, or 19.

[0034] Sequence identity Sequence identity can be calculated using any suitable algorithm. For example, the PILEUP and BLAST algorithms can be used to calculate identity and align sequences, such as identifying equivalent or corresponding sequences (typically in their initial settings), as described, for example, by Altschul SF (1993), J Mol Evol, vol. 36: pp. 290-300; and Altschul, S, F et al. (1990), J Mol Biol, vol. 215: pp. 403-4010. Software for performing BLAST analysis is available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying shorter words in the query sequence that, when aligned with words of the same length in the database sequences, match or satisfy a threshold score T of a certain positive value. T is called the neighbor word score threshold (Altschul et al., see above). These initial neighbor word hits act as seeds to initiate the search for HSPs containing them. These word hits are extended bidirectionally along each sequence as long as they can increase the cumulative alignment score.

[0035] The extension of word hits in each direction is stopped if the cumulative alignment score decreases by X from its maximum attainable value; if the cumulative score becomes 0 or less due to the accumulation of one or more negative score residue alignments; or if the end of any sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. By default, the BLAST program uses word length (W)=11, BLOSUM62 score matrix (see Henikoff and Henikoff (1992), Proc. Natl. Acad. Sci. USA, Vol. 89: pp. 10915-10919), alignment (B)=50, expected value (E)=10, M=5, N=4, and quantitative comparison.

[0036] The BLAST algorithm performs statistical analysis of the similarity between two sequences; see, for example, Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 5873-5787.

[0037] One similarity measure provided by the BLAST algorithm is the smallest sum probability (P(N)), which is an indicator of the probability that a match occurs by chance between two polynucleotide sequences or two amino acid sequences. For example, if the smallest sum probability when comparing the first sequence to the second sequence is less than approximately 1, then one sequence is considered similar to the other; less than approximately 0.1 is preferable, less than approximately 0.01 is more preferable, and less than approximately 0.001 is most preferable. Alternatively, the UWGCG package provides a BESTFIT program that can be used to calculate identity (e.g., used in the default settings) (Devereux et al. (1984), Nucleic Acids Research, Vol. 12, pp. 387-395).

[0038] nucleic acid The present invention further provides nucleic acids encoding the modified AAV capsid protein of the present invention. In some embodiments, the nucleic acids of the present invention include the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 9, or a 21-nucleotide insertion sequence encoding a variant thereof as described above. In one exemplary embodiment, the nucleic acids of the present invention include the nucleotide sequence of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, or 37. In further exemplary embodiments, the nucleic acids of the present invention include a 21-nucleotide insertion sequence encoding a variant of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 9. Examples of such sequences include SEQ ID NOs: 301-304, 316-318, and 330-335, which retain the ability to encode the modified AAV capsid protein of the present invention and form viral particles that infect and / or transduce microglia or brain macrophages. In situations involving infection and / or transduction of microglia or cerebral macrophages, the nucleic acids of the present invention may contain any of the nucleotide sequences of the insertion sequences (SEQ ID NOs. 208-361) shown in Table 4. In some embodiments, the nucleic acids of the present invention include at least 21-nucleotide insertion sequences, at least 24-nucleotide insertion sequences, at least 27-nucleotide insertion sequences, or at least 30-nucleotide insertion sequences encoding the amino acid sequence of SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, or 9, or a variant thereof as described above.

[0039] In a preferred embodiment of the present invention, the nucleic acid has the nucleotide sequence of SEQ ID NOs. 20, 21, 22, 23, 24, 25, 26, 27, or 28. The nucleic acid of the present invention also includes variants of SEQ ID NOs. 20, 21, 22, 23, 24, 25, 26, 27, or 28 that have a different sequence but retain the ability to encode the modified AAV capsid protein of the present invention. Such sequences have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NOs. 20, 21, 22, 23, 24, 25, 26, 27, or 28. For example, the nucleic acid may be modified to replace the nucleotide sequence of SEQ ID NOs. 29, 30, 31, 32, 33, 34, 35, 36, or 37 with one of the nucleotide sequences of the insertion sequences (SEQ ID NOs. 208 to 361) shown in Table 4.

[0040] The variant sequence identity percentage is preferably measured over the full length of the corresponding portion of SEQ ID NOs. 20, 21, 22, 23, 24, 25, 26, 27, or 28 aligned with the variant sequence, or over a section of 500, 1000, 1500, or 2000 consecutive amino acids in SEQ ID NOs. 20, 21, 22, 23, 24, 25, 26, 27, or 28.

[0041] As is known to those skilled in the art, amino acid residues may be similarly encoded by multiple nucleotide sequences. Therefore, in one embodiment, the present invention encompasses degenerate nucleic acids whose codon sequences differ from sequence numbers 20, 21, 22, 23, 24, 25, 26, 27, or 28 due to the degeneracy of the genetic code.

[0042] The present invention further provides recombinant DNA comprising the nucleic acid of the present invention or a variant thereof as described above. The recombinant DNA may be a recombinant AAV vector comprising the nucleic acid of the present invention or a variant thereof as described above. Thus, such a recombinant AAV vector encodes the modified AAV capsid protein of the present invention.

[0043] Typically, by incorporating all or part of the nucleic acid of the present invention into an AAV genome, the AAV genome can be made to encode the modified capsid protein of the present invention. Therefore, such an AAV genome may contain the nucleic acid sequence of SEQ ID NOs. 20, 21, 22, 23, 24, 25, 26, 27, or 28, or a variant thereof as described above.

[0044] The AAV genome of the present invention can be obtained from an AAV having any serotype. In some embodiments, the AAV genome of the present invention is obtained from a serotype different from that of the capsid protein of the present invention. Preferably, the AAV genome is obtained from AAV serotype 2 (AAV2), and the modified capsid protein is obtained from AAV9.

[0045] Such AAV genomes may additionally encode functions necessary for the production of AAV virus particles in host cells. This allows for the production of AAV virus particles incorporating the modified AAV capsid protein of the present invention by using the AAV genome of the present invention. Naturally occurring AAV viruses are replication-deficient and rely on the trans to provide helper functions to complete the replication and packaging cycle. Therefore, such AAV virus particles may be replication-deficient.

[0046] Virus particles As described above, the present invention further provides AAV virus particles. The AAV particles have a shell composed of many capsid proteins. Typically, the AAV virus particles of the present invention are equipped with the modified capsid protein of the present invention. Thus, in some embodiments, the AAV particles of the present invention include the modified AAV capsid protein of the present invention having the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 9, or variants thereof as described above. Examples of such variants include SEQ ID NOs: 147-150, 162-164, and 176-181, which retain the ability to form AAV virus particles that infect and / or transduce microglia or cerebral macrophages. In the context of infecting and / or transduction of microglia or cerebral macrophages, the AAV virus particles of the present invention may include the modified AAV capsid protein having the amino acid sequence of any of the insertion sequences (SEQ ID NOs: 54-207) shown in Table 4. In some embodiments, the AAV virus particles of the present invention may optionally carry a cargo that can be delivered to cells. In a preferred embodiment, the AAV virus particles of the present invention comprise the modified capsid protein and AAV genome of the present invention. In one embodiment, the AAV particle comprises an AAV genome containing two ITRs. The AAV virus particles of the present invention encompass a capsid-converted form in which an AAV genome or derivative having one serotype ITR is packaged in the AAV capsid protein of the present invention. In one embodiment, the AAV particle of the present invention comprising the modified capsid protein of the present invention may carry an AAV genome encoding the modified AAV capsid protein of the present invention. Such AAV virus particles are capable of delivering the viral genome to cells. In embodiments of the present invention, the cells are nerve cells. In embodiments of the present invention, the cells are glial cells, oligodendrocytes, or astrocytes. In another embodiment of the present invention, the cells are microglia, or resident macrophages including recruited monocyte-derived CNS-infiltrating macrophages.In a preferred embodiment of the present invention, the AAV virus particles of the present invention deliver the AAV genome to microglia or brain macrophages.

[0047] In one embodiment, the AAV particles of the present invention comprise the modified AAV capsid protein of the present invention and further comprise recombinant polynucleotides encoding a target gene, a gene editing construct, an antibody or antigen-binding fragment, or a gene silencing construct for delivery to a cell. The present invention further provides a host cell containing the AAV virus particles of the present invention.

[0048] The AAV virus particles of the present invention may also include chemically modified forms that carry ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting specific cell surface receptors.

[0049] Implementation efficiency Viral particles carrying the AAV capsid protein of the present invention exhibit increased delivery efficiency in cells compared to viral particles carrying wild-type AAV capsid protein or AAV capsid protein without the amino acid sequence insertion of the present invention. In exemplary embodiments, viral particles having the AAV capsid protein of the present invention exhibit increased delivery efficiency in microglia or brain macrophages compared to viral particles carrying wild-type AAV capsid protein or AAV capsid protein without the amino acid sequence insertion of the present invention. For example, viral particles having the AAV capsid protein of the present invention exhibit increased delivery efficiency in microglia compared to viral particles having unmodified AAV capsid protein having the sequence of SEQ ID NO: 10.

[0050] The delivery efficiency can be analyzed by any suitable standard technique known to those skilled in the art, for example, using fluorescent labeling. Viral particles having the AAV capsid protein of the present invention may show at least a 2-fold, at least a 5-fold, at least a 10-fold, at least a 20-fold, or at least a 50-fold increase in delivery efficiency in cells compared to viral particles having wild-type AAV capsid protein or AAV capsid protein without the amino acid sequence insertion of the present invention. Viral particles having the AAV capsid protein of the present invention may show at least a 1%, 5%, 10%, 20%, 40%, 80%, 90%, 95%, or 99% increase in delivery efficiency in cells compared to viral particles having wild-type AAV capsid protein or AAV capsid protein without the amino acid sequence insertion of the present invention.

[0051] host cell The present invention further provides a host cell comprising the nucleic acid or recombinant DNA disclosed herein. The nucleic acid or recombinant DNA may be provided as an episomal component such as a plasmid within the host cell, or one or more constructs may be incorporated into the genome of the host cell.

[0052] The host cells of the present invention are capable of producing the viral particles of the present invention. In order to enable the construction of a derivatized genome within the AAV viral particles, additional genetic constructs conferring AAV function and / or helper virus function are provided to the host cells in combination with the derivatized genome.

[0053] Any suitable host cell can be used to produce the AAV virus particles of the present invention. Typically, such cells are transfected mammalian cells, but other cell types, such as insect cells, can also be used. In mammalian cell production systems, HEK293 and HEK293T are preferred for the AAV vector. BHK cells or CHO cells may also be used.

[0054] Use of modified AAV virus particles As described above, the AAV virus particles of the present invention can be used to deliver cargo to cells. In some embodiments, the AAV virus particles of the present invention, comprising the modified AAV capsid protein of the present invention, can be used to deliver nucleic acids and / or the AAV genome to cells. In embodiments of the present invention, the cells are nerve cells. In embodiments of the present invention, the cells are glial cells, oligodendrocytes, or astrocytes. In another embodiment of the present invention, the cells are microglia, or resident brain macrophages, including recruited monocyte-derived CNS-infiltrating macrophages. In preferred embodiments of the present invention, the cells are microglia or intracerebral macrophages. In exemplary embodiments, the AAV capsid protein of the present invention can target microglia. That is, the present invention encompasses the use of any one of SEQ ID NOs: 1-9, or its variants having at least one amino acid substitution, at least two amino acid substitutions, or at least three amino acid substitutions, in targeting and / or infection of microglia. For example, the present invention includes the use of any one of the insertion sequences (SEQ ID NOs. 54-207) shown in Table 4 for targeting and / or infection of microglia or cerebral macrophages.

[0055] In one embodiment, the AAV virus particle of the present invention, comprising the modified AAV capsid protein of the present invention, optionally comprises a recombinant polynucleotide encoding a target gene, a gene editing construct, an antibody or antigen-binding fragment, or a gene silencing construct, and preferably incorporated into the AAV genome within the particle. In a preferred embodiment, the AAV virus particle of the present invention comprises a recombinant polynucleotide encoding a target gene, a gene editing construct, an antibody or antigen-binding fragment, or a gene silencing construct, and comprises an AAV genome encoding the modified capsid protein of the present invention.

[0056] In some embodiments, the AAV virus particles of the present invention can be used in gene therapy. For example, target cells such as microglia cells transduced with the AAV virus particles of the present invention containing recombinant polynucleotides can express target genes, construct targets for gene editing, binding targets for antibodies or antigen-binding fragments, or targets for gene silencing.

[0057] Any gene expressed in brain cells, particularly microglia, and involved in neurological diseases can be considered a target gene. For example, target genes may be involved in neurodegenerative diseases such as Alzheimer's disease; neuroinflammatory diseases such as multiple sclerosis; neurodevelopmental disorders such as autism; neuropsychiatric disorders such as schizophrenia; seizure-related disorders such as epilepsy; cerebrovascular diseases such as stroke; brain cancers such as gliablastoma; motor disorders such as Parkinson's disease; neuroinfectious diseases such as encephalitis; pain-related disorders such as migraines; or acute brain injuries such as traumatic brain injury. Non-restrictive and exemplary target genes include TREM2 in relation to phagocytosis, CCL4 / CCL3 in relation to cell migration, CD22 in relation to cell rejuvenation, and CSF1R in relation to cell survival. The most detailed examples of genes related to microglial function in development and disease are given (Keren-Shaul et al., 2017; Young et al., 2019; Schirmer et al., 2019; Hammond et al., 2019; Masuda et al., 2019; Giersdottir et al., 2019).

[0058] In embodiments of the present invention, the AAV virus particles of the present invention optionally include an AAV genome incorporating a recombinant polynucleotide encoding a target gene. The target gene may be associated with neurodegenerative diseases such as Alzheimer's disease, neurodevelopmental disorders, psychosomatic schizophrenia, or acute brain injury. Non-restrictive and exemplary target genes of interest include TREM2, CCL4 / CCL3, CD22, and CSF1R. Thus, the AAV virus particles of the present invention can be used to significantly increase gene expression in brain cells, particularly microglia. Significant increase in expression can be defined as an increase of approximately 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, or 300-fold in the expression of TREM2, CCL4 / CCL3, CD22, and CSF1R in cells compared to wild-type expression of TREM2, CCL4 / CCL3, CD22, and CSF1R. The expression levels of TREM2, CCL4 / CCL3, CD22, and CSF1R can be measured using any suitable standard technique known to those skilled in the art. For example, RNA expression levels can be measured by quantitative real-time PCR. Protein expression can be measured by Western blotting or immunohistochemistry.

[0059] In embodiments of the present invention, the AAV virus particles of the present invention optionally include an AAV genome incorporating recombinant polynucleotides encoding gene-editing constructs used to edit target gene sequences in brain cells, particularly microglia. These genes may be associated with neurodegenerative diseases such as Alzheimer's disease, neurodevelopmental disorders, psychosomatic schizophrenia, or acute brain injury. Non-restrictive and exemplary target genes for gene editing include TREM2, CCL4 / CCL3, CD22, and CSF1R.

[0060] The present invention encompasses various mechanisms for performing gene editing using the AAV virus particles of the present invention. Such mechanisms include endonuclease-based gene editing methods, which include, but are not limited to, zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), meganucleases (such as MegaTAL), and CRISPR / Cas9. In some embodiments, the AAV virus particles of the present invention optionally include an AAV genome incorporating a CRISPR guide RNA and a recombinant polynucleotide encoding the Cas9 protein or its derivatives or fragments. In preferred embodiments, the AAV virus particles of the present invention optionally include an AAV genome incorporating a CRISPR guide RNA complementary to TREM2, CCL4, CCL3, CD22, or CSF1R, and a recombinant polynucleotide encoding the Cas9 protein or its derivatives or fragments.

[0061] In embodiments of the present invention, the AAV virus particles of the present invention optionally include an AAV genome incorporating recombinant polynucleotides encoding antibodies or antigen-binding fragments that bind to gene products in brain cells, particularly microglia. These genes may be associated with neurodegenerative diseases such as Alzheimer's disease, neurodevelopmental disorders, psychosomatic schizophrenia, or acute brain injury. Non-limiting and exemplary target genes for the binding of antibodies or antigen-binding fragments include TREM2, CCL4 / CCL3, CD22, and CSF1R. Therefore, the AAV virus particles of the present invention can be used in gene replacement therapy applications.

[0062] As used herein, the term “antibody” encompasses immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as their polymers (e.g., IgM). As used herein, the term “antibody” also encompasses antigen-binding fragments of complete antibody molecules, or any derivatives thereof, that contribute to the formation of a “functional antibody” and may exhibit desired biological activity. As used herein, terms such as “antigen-binding moiety” of an antibody and “antigen-binding fragment” of an antibody encompass any naturally occurring, synthetically obtained, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0063] Non-limiting examples of antigen-binding fragments include (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv(scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking antibodies (e.g., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide) or the hypervariable region of restrictive FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other modified molecules such as shark variable IgNAR domains are also included in the expression “antigen-binding fragment” as used herein.

[0064] As used herein, the term "antibody" also includes multispecific (e.g., bispecific) antibodies. A multispecific antibody or antigen-binding fragment of an antibody typically comprises at least two distinct variable domains, each of which is capable of specifically binding to a different antigen or to a different epitope on the same antigen.

[0065] In embodiments of the present invention, the AAV virus particles of the present invention optionally include an AAV genome incorporating recombinant polynucleotides encoding gene silencing constructs used to silence the expression and / or activity of target genes in brain cells, particularly microglia. These genes may be associated with neurodegenerative diseases such as Alzheimer's disease, or with neurodevelopmental disorders, psychosomatic schizophrenia, or acute brain injury. Non-restrictive and exemplary target genes for gene silencing include TREM2, CCL4 / CCL3, CD22, and CSF1R.

[0066] Various mechanisms for silencing gene expression or activity using the AAV virus particles of the present invention are included in this invention.

[0067] As used herein, the term "silencing" encompasses reduction, inhibition, or downregulation of gene expression, reduction, inhibition, or downregulation of transcription, reduction, inhibition, or downregulation of translation, and / or reduction, inhibition, or downregulation of protein activity. Such reduction, inhibition, or downregulation may be direct or indirect. Methods for measuring the level of reduction, inhibition, or downregulation of gene expression, reduction, inhibition, or downregulation of transcription, reduction, inhibition, or downregulation of translation, and / or reduction, inhibition, or downregulation of protein activity are known to those skilled in the art. Examples include insight hybridization for measuring gene expression and immunoblotting for measuring protein expression. Such reduction, inhibition, or downregulation may be complete or partial. The silencing described herein can be considered to encompass a 10% reduction, inhibition, or downregulation of gene expression, transcription, translation, and / or protein activity, including 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction, inhibition, or downregulation. The level of inhibition of gene expression can be measured by those skilled in the art using methods known in the art.

[0068] In some embodiments of the present invention, recombinant polynucleotides may include sequences that act to silence gene expression, transcription, translation and / or protein activity. In such embodiments of the present invention, recombinant polynucleotides may include double-stranded RNA, ncRNA, shRNA, siRNA, miRNA, CRISPR enzyme sequences (such as Cas-9, dCas-9, SaCas-9, dSaCas-9, dSaCas-9-KRAB), guide RNA, zinc finger proteins (ZFPs), transcription activator-like effector nucleases (TALENs) and / or DREADD.

[0069] double stranded RNA Using known techniques and based on knowledge of the sequence of the gene to be silenced, a double-stranded RNA (dsRNA) molecule can be designed to silence the gene by targeting based on the sequence homology of the gene's RNA. Such dsRNAs are typically small interfering RNAs (siRNAs) or microRNAs (miRNAs) with a stem-loop ("hairpin") structure. The sequence of such a dsRNA will include a portion that matches the sequence of a part of the mRNA encoding the gene. This portion is usually 100% complementary to the target region in the gene's mRNA, but lower levels of complementarity (e.g., 90% or more, or 95% or more) can also be used.

[0070] siRNA In one embodiment, the silencing mechanism includes a small interfering RNA (siRNA). The siRNA acts by activating an RNAi-induced repression complex. The siRNA molecule may be unmodified or modified and is capable of repressing gene expression. The siRNA molecule is typically about 15–60 nucleotides long. In some embodiments, the modified siRNA contains at least one 2'O-Me purine nucleotide or 2'O-Me pyrimidine nucleotide, such as a 2'O-Me-guanosine nucleotide, a 2'O-Me-uridine nucleotide, a 2'O-Me-adenosine nucleotide, and / or a 2'O-Me-cytosine nucleotide. The modified nucleotide may be present on one strand of the siRNA (i.e., the sense strand or the antisense strand) or on both strands. The siRNA sequence may have overhangs or blunt ends.

[0071] Modified siRNA may contain approximately 1% to 100% (for example, approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the double-stranded region of the siRNA double strand. In one embodiment, one, two, three, four, five, six, seven, eight, nine, ten, or more nucleotides in the double-stranded region of the siRNA contain modified nucleotides.

[0072] Suitable siRNA sequences can be identified using any means known in the art. Typically, the methods described in Elbashir et al., Nature, Vol. 411: pp. 494-498 (2001) and Elbashir et al., EMBO J., Vol. 20: pp. 6877-6888 (2001) are combined with the rational design rules described in Reynolds et al., Nature Biotech., Vol. 22 (No. 3): pp. 326-330 (2004).

[0073] siRNA is preferably chemically synthesized. Oligonucleotides containing the siRNA molecule of the present invention can be synthesized using any of the various methods known in the art, such as those described in Usman et al., J.Am.Chem.Soc., Vol. 109:p7845 (1987); Scaringe et al., Nucl. Acids Res., Vol. 18:p5433 (1990); Wincott et al., Nucl. Acids Res., Vol. 23:pp2677~2684 (1995); and Wincott et al., Methods Mol. Bio., Vol. 74:p59 (1997). The synthesis of oligonucleotides utilizes common nucleic acid protecting groups and coupling groups, such as dimethoxytrityl at the 5' end and phosphoramidite at the 3' end. Alternatively, the siRNA molecule can be assembled from two separate oligonucleotides, one containing the sense strand and the other containing the siRNA antisense strand. For example, each strand can be synthesized separately and then joined by hybridization or ligation after synthesis and / or deprotection. In certain other cases, the siRNA molecule may be synthesized as a single, continuous oligonucleotide fragment in which self-complementary sense and antisense regions hybridize to form a double-stranded siRNA having a hairpin secondary structure.

[0074] CRISPR and guide RNA In one embodiment, the silencing mechanism encompasses a gene silencing mechanism using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). In one embodiment, the gene silencing mechanism using CRISPR involves the use of guide RNA. The guide RNA may include a guide RNA sequence and a transactivating crRNA (tracrRNA). The guide RNA sequence can hybridize to a target sequence in the DNA to be silenced. The tracrRNA is ligated to the guide RNA sequence. The guide RNA hybridizes to an allele site, targeting the CRISPR-Cas enzyme to that site.

[0075] In some embodiments, the guide RNA is 10–30 nucleotides long, or 15–25 nucleotides long, or 15–20 nucleotides long. In some embodiments, one guide RNA is used. In some embodiments, two guide RNAs are used. In some embodiments, three or more guide RNAs are used.

[0076] The CRISPR-Cas enzyme is preferably a type II CRISPR enzyme, such as Cas-9 (CRISPR-related protein 9). In some preferred embodiments, the Cas-9 enzyme is SaCas-9.

[0077] The enzyme forms a complex with a guide RNA. In one embodiment, the complex, targeted to a DNA sequence, binds by hybridization. In one embodiment, the enzyme acts as an endonuclease, cleaving DNA via non-homologous end joining or activation of homologous DNA repair pathways, resulting in blunt end breaks or nicks. In one embodiment, using one or more guide RNAs and a CRISPR enzyme, essential components of the gene to be silenced are deleted, resulting in a non-functional gene. In some embodiments, the gene is not transcribed. In some embodiments, the gene is not translated.

[0078] In another embodiment, the enzyme is targeted to the DNA of the gene to be silenced, but the enzyme does not edit the mutant allele, but rather prevents or reduces its transcription, by including one or more mutations that reduce or eliminate its endonuclease activity. An example of such an enzyme for use in the present invention is dCas-9, which is an enzyme that does not exhibit catalytic activity. In a preferred embodiment, dCas-9 is dSa-Cas9.

[0079] In one embodiment, dCas-9 is associated with a transcriptional repressor peptide that can knock down gene expression by interfering with transcription. In a preferred embodiment, the transcriptional repressor protein is a Kruppel-associated box (KRAB).

[0080] In related embodiments, the enzyme may be designed to be fused with a transcriptional repressor to reduce or disable its endonuclease function. The enzyme is capable of binding to guide RNA and targeting a DNA sequence, but without cleaving the DNA. The mutant allele can be repressed, for example, by shutting down the promoter or blocking the RNA polymerase.

[0081] In another embodiment, the transcriptional repressor may be bound to the tracr sequence. As described by Konermann et al. (Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex, Nature, Vol. 000, 2014), the functional domain can be attached to the tracr sequence by incorporating a protein-binding RNA aptamer sequence. The transcriptional repressor-tracr sequence complex can be used to target other substructures to desired precise gene locations.

[0082] In another embodiment, the CRISPR silencing mechanism includes a CRISPR-based editor for knocking out a gene by altering a single nucleotide to form a stop codon (CRISPR-STOP method (Kuscu et al., 2017)).

[0083] In another embodiment, the CRISPR silencing mechanism includes gene upregulation via CRISPR activation, resulting in the silencing of a target gene as described herein.

[0084] ZFP In another embodiment of the present invention, the silencing mechanism involves the use of a zinc finger protein (ZFP, also known as zinc finger nuclease or ZFN). A ZFP is a heterodimer in which each subunit contains a zinc finger domain and a FokI endonuclease domain. ZFPs constitute the largest individual family of transcriptional regulators known in higher organisms.

[0085] TALEN In another embodiment of the present invention, the silencing mechanism includes the use of a transcription activator-like effector nuclease (TALEN). The TALEN comprises a nonspecific DNA cleavage nuclease fused to a customizable DNA-binding domain so that the TALEN can target a target sequence to be silenced (Joung and Sander, 2013).

[0086] DREADD In another embodiment of the present invention, the silencing mechanism involves the use of a designer receptor exclusively activated by designer drugs (DREADD). DREADD is a family of designer G protein-coupled receptors (GPCRs) specifically constructed to enable precise spatiotemporal control of GPCR signaling in vivo, which controls neuronal excitability. DREADD systems have been used to selectively inhibit or activate the electrical activity of neurons (Magnus et al., 2019).

[0087] Promoter and enhancer for use in delivery according to the present invention In one embodiment, the viral genome of an AAV particle as described herein includes at least one regulatory element that enables replication, transcription, and translation of the coding sequence encoded in the viral genome. In a preferred embodiment of the present invention, the at least one regulatory element is a cell type-specific promoter and / or enhancer.

[0088] In another related embodiment, the viral genome contained in the AAV virus particle of the present invention, which includes a coding region encoding a gene or interest or a gene silencing construct, further includes a microglia-specific promoter and / or enhancer, or a macrophage-specific promoter and / or enhancer. The target gene or gene silencing construct is typically functionally linked to the promoter and / or enhancer. The promoter and / or enhancer may be constitutive, but is preferably a microglia-specific or infiltrating brain macrophage-specific promoter and / or enhancer.

[0089] Microglia-specific promoters and / or enhancers mean promoters and / or enhancers that preferentially drive expression in microglial cells, or drive expression in microglial cells only or substantially only in microglial cells, for example, promoters and / or enhancers that drive expression at least twice, at least five times, at least ten times, at least twenty times, or at least fifty times more strongly in microglia than in any other cell type.

[0090] Infiltrating brain macrophage-specific promoters and / or enhancers mean promoters and / or enhancers that preferentially drive expression in infiltrating brain macrophages, or that drive expression in infiltrating brain macrophages only or substantially only in infiltrating brain macrophages, for example, promoters and / or enhancers that drive expression at least 2 times, at least 5 times, at least 10 times, at least 20 times, or at least 50 times more strongly in infiltrating brain macrophages than in any other cell type. In preferred embodiments, the microglia-specific promoter is a promoter derived from TMEM119, CX3CR1, or P2Y12 (P2RY12). In preferred embodiments, the macrophage-specific promoter is a promoter derived from CD11b, CD68, CSF1R, or F4 / 80. A list of microglia-related genes is provided (Keren-Shaul et al., 2017; Young et al., 2019; Schirmer et al., 2019; Hammond et al., 2019; Masuda et al., 2019; Giersdottir et al., 2019). In some embodiments, the promoter drives expression in microglia and infiltrating brain macrophages. In one embodiment, the promoter may be a macrophage-specific promoter that drives expression in microglia. For example, the viral genome contained in the AAV virus particle of the present invention, which includes a coding region encoding a gene or interest or a construct for gene silencing, further comprises the macrophage-specific promoter CD11b.

[0091] In another related embodiment, the viral genome contained in the AAV virus particle of the present invention, including a coding region encoding a gene or interest or a gene silencing construct, further includes localization sequences for further restricting gene expression to separate intracellular compartments such as the nucleus, cell membrane, or cytosol. In an exemplary embodiment, the viral genome contained in the AAV virus particle of the present invention, including a coding region encoding a target gene or a gene silencing construct, further includes a nuclear localization signal (NLS), a nuclear exclusion signal (NES), or a membrane targeting signal.

[0092] Non-human transgenic animals The present invention further provides a transgenic animal comprising cells containing the capsid protein or viral particles of the present invention. The animal is preferably a mammal other than a human, particularly a primate. Alternatively, the animal may be a rodent, particularly a mouse; or it may be a dog, cat, sheep, or pig.

[0093] Pharmaceutical composition and dosage The AAV capsid protein, nucleic acid, or viral particles of the present invention can be formulated into pharmaceutical compositions. These compositions may contain, in addition to the AAV capsid protein, nucleic acid, or viral particles, pharmaceutically acceptable excipients, carriers, diluents, buffers, adjuvants, stabilizers, and / or other substances well known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact properties of carriers or other substances can be determined by those skilled in the art based on the route of administration.

[0094] The above-mentioned pharmaceutical compositions are typically liquid. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may also contain physiological saline, magnesium chloride, a sugar solution such as glucose, or a glycol such as ethylene glycol, propylene glycol, or polyethylene glycol. In some cases, a surfactant such as 0.001% pluronic acid (PF68) may be used.

[0095] For injection into the affected area, the active ingredient will be in the form of an aqueous solution that is pyrogenic and has a suitable pH, isotonicity, and stability. Those skilled in the art will have ample ability to prepare a suitable solution using an isotonic medium such as sodium chloride injection, Ringer's solution, lactated Ringer's solution, or Hartmann's infusion. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0096] For delayed release, the AAV capsid protein, nucleic acid, or viral particles may be contained in a pharmaceutical composition prepared for sustained release, such as in a microcapsule formed from a biocompatible polymer or in a liposome carrier system, according to methods known in the art.

[0097] The dosage regime may be determined within the scope of the physician's usual skill in administering the composition. The dose of the active agent may vary depending on the reason for use, the individual subject, and the mode of administration. The dose may be adjusted based on the subject's body weight, age and health, as well as their tolerance to the compound or composition.

[0098] Therapeutic methods and medical uses The AAV virus particles of the present invention may be used in the treatment of a subject. The terms “patient” and “subject” may be used synonymously. The patient is preferably a mammal. The mammal may be a commercially farmed animal such as a horse, cattle, sheep, or pig, a laboratory animal such as a mouse or rat, or a pet such as a cat, dog, rabbit, or guinea pig. More preferably, the patient is a human. The subject may be male or female.

[0099] The subjects are preferably identified as being at risk of or having a neurological disorder. Exemplary classifications and examples include, but are not limited to, the following: neurodegenerative diseases such as Alzheimer's disease; neuroinflammatory diseases such as multiple sclerosis; neurodevelopmental disorders such as autism; neuropsychiatric disorders such as schizophrenia; seizure-related disorders such as epilepsy; cerebrovascular diseases such as stroke; brain cancers such as gliablastoma; motor disorders such as Parkinson's disease; neuroinfectious diseases such as encephalitis; pain-related disorders such as migraines; or acute brain injuries such as traumatic brain injury.

[0100] The terms “treat,” “treated,” “treating,” or “treatment,” as used herein, refer to both therapeutic and preventive or deterrent actions aimed at preventing or delaying (reducing) an undesirable physiological condition, disorder, or disease, or at obtaining a beneficial or desirable clinical outcome. Beneficial or desirable clinical outcomes include, but are not limited to, relief of symptoms; reduction of the severity of a condition, disorder, or disease; stabilization of the condition, disorder, or disease (i.e., no exacerbations); delay of the onset or slowing of the progression of a condition, disorder, or disease; improvement of the condition, disorder, or disease; and remission (including partial or complete remission), improvement, or improvement of a condition, disorder, or disease, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without excessive levels of adverse effects.

[0101] The AAV virus particles of the present invention may be used in the treatment or prevention of neurodegenerative diseases, neuroinflammatory diseases, neurodevelopmental disorders, neuropsychiatric disorders, seizure-related disorders, cerebrovascular diseases, brain cancer, motor disorders, neuroinfections, pain-related disorders, or acute brain injury. This provides a means to treat, halt, mitigate, or prevent the degenerative processes of the above diseases.

[0102] Based on the above, the present invention provides a pharmaceutical composition comprising the AAV virus particles of the present invention and a pharmaceutically acceptable carrier.

[0103] Furthermore, the present invention provides AAV virus particles for use in methods of preventing or treating neurodegenerative diseases, neuroinflammatory diseases, neurodevelopmental disorders, neuropsychiatric disorders, seizure-related disorders, cerebrovascular diseases, brain cancer, motor disorders, neuroinfections, pain-related disorders, or acute brain injury.

[0104] Furthermore, the present invention provides for the use of AAV virus particles in the manufacture of pharmaceuticals for the treatment or prevention of neurodegenerative diseases, neuroinflammatory diseases, neurodevelopmental disorders, neuropsychiatric disorders, seizure-related disorders, cerebrovascular diseases, brain cancer, motor disorders, neuroinfections, pain-related disorders, or acute brain injury.

[0105] Furthermore, the present invention provides a method for treating or preventing neurodegenerative diseases, neuroinflammatory diseases, neurodevelopmental disorders, neuropsychiatric disorders, seizure-related disorders, cerebrovascular diseases, brain cancer, motor disorders, neuroinfections, pain-related disorders, or acute brain injury in patients requiring treatment, comprising administering a therapeutically effective amount of the AAV virus particles of the present invention to a patient.

[0106] By using the AAV virus particles of the present invention, which contain the modified AAV capsid protein of the present invention, the expression of a target gene, gene editing construct, antibody or antigen-binding fragment, or gene silencing construct can be achieved throughout CNS cells, particularly in microglia and infiltrating intracerebral macrophages. Therefore, the AAV virus particles of the present invention can be used to treat or prevent neurodegenerative diseases, neuroinflammatory diseases, neurodevelopmental disorders, neuropsychiatric disorders, seizure-related disorders, cerebrovascular diseases, brain cancer, motor disorders, neuroinfections, pain-related disorders, or acute brain injury by treating the microglia and infiltrating intracerebral macrophages of such patients by expressing a target gene, gene editing construct, antibody or antigen-binding fragment, or gene silencing construct throughout CNS cells, particularly in microglia and infiltrating intracerebral macrophages.

[0107] Parenteral delivery routes of the AAV of the present invention, such as intravenous (IV) or intraventricular (ICV) administration, typically by injection or infusion, are usually preferred.

[0108] Accordingly, the present invention also provides a method for treating or preventing neurodegenerative diseases, neuroinflammatory diseases, neurodevelopmental disorders, neuropsychiatric disorders, seizure-related disorders, cerebrovascular diseases, brain cancer, motor disorders, neuroinfections, pain-related disorders, or acute brain injury in patients requiring treatment, comprising administering a therapeutically effective amount of the AAV virus particles of the present invention to a patient via a parenteral route. Thus, in the above patients, neurodegenerative diseases, neuroinflammatory diseases, neurodevelopmental disorders, neuropsychiatric disorders, seizure-related disorders, cerebrovascular diseases, brain cancer, motor disorders, neuroinfections, pain-related disorders, or acute brain injury are thereby treated or prevented.

[0109] In a related embodiment, the present invention provides the use of AAV virus particles in a method of treating or preventing neurodegenerative diseases, neuroinflammatory diseases, neurodevelopmental disorders, neuropsychiatric disorders, seizure-related disorders, cerebrovascular diseases, brain cancer, motor disorders, neuroinfections, pain-related disorders, or acute brain injury by administering AAV virus particles to a patient via a parenteral administration route. Furthermore, the present invention provides the use of AAV virus particles in the manufacture of a pharmaceutical product for treating or preventing neurodegenerative diseases, neuroinflammatory diseases, neurodevelopmental disorders, neuropsychiatric disorders, seizure-related disorders, cerebrovascular diseases, brain cancer, motor disorders, neuroinfections, pain-related disorders, or acute brain injury via a parenteral administration route.

[0110] In all of these embodiments, the AAV virus particles of the present invention may be administered for the purpose of preventing the development of one or more symptoms of neurodegenerative disease, neuroinflammatory disease, neurodevelopmental disorder, neuropsychiatric disorder, seizure-related disorder, cerebrovascular disease, brain cancer, motor disorder, neuroinfection, pain-related disorder, or acute brain injury. The patient may be asymptomatic. The subject may have a predisposition to the above diseases. The above method or use may include a step of determining whether or not the subject is at risk of developing, or has risk of developing, a neurodegenerative disease, neuroinflammatory disease, neurodevelopmental disorder, neuropsychiatric disorder, seizure-related disorder, cerebrovascular disease, brain cancer, motor disorder, neuroinfection, pain-related disorder, or acute brain injury. A prophylactic effective dose of AAV is administered to such subject. The prophylactic effective dose is a dose that prevents the development of one or more symptoms of the disease.

[0111] Alternatively, the AAV virus particles of the present invention may be administered after the symptoms of the disease have appeared in the subject, i.e., to treat pre-existing disease symptoms. A therapeutically effective dose of the above antagonist is administered to such subject. The therapeutically effective dose is an amount effective in improving one or more disease symptoms.

[0112] The subjects may be male or female. Preferably, the subjects are identified as being at risk of disease or having a disease.

[0113] The administration of the AAV viral particles of the present invention is typically by a parenteral route of administration. Parenteral routes of administration include intravenous (IV), intramuscular (IM), subcutaneous (SC), epidural (E), intracerebral (IC), intraventricular (ICV), intranasal (IN), and intradermal (ID) administrations.

[0114] The single-dose amount of the AAV viral particles of the present invention may be determined according to various parameters, particularly the age, weight and condition of the patient to be treated; the route of administration; and the required dosing schedule. As before, a physician can determine the route of administration and dosage required for any particular patient. For example, a suitable single-dose amount of AAV of the present invention may be in the range of about 1×10 6 vg to about 1×10 16 vg, where vg is the viral genome. In some embodiments, suitable single-dose amounts of AAV of the present invention are about 1×10 6 vg, about 1×10 7 vg, about 1×10 8 vg, about 1×10 9 vg, about 1×10 10 vg, about 1×10 11 vg, about 1×10 12 vg, about 1×10 13 vg, about 1×10 14 vg, about 1×10 15 vg, or about 1×10 16 vg.

[0115] Any suitable dosing schedule for administering the AAV of the present invention may be used, including single-dose regimens or multiple-dose regimens such as divided-dose regimens.

[0116] Combination therapy The capsid protein, nucleic acid, viral particle and / or pharmaceutical composition can be used in combination with any other therapy for the treatment or prevention of neurodegenerative diseases, neuroinflammatory diseases, neurodevelopmental disorders, neuropsychiatric disorders, seizure-related disorders, cerebrovascular-related diseases, brain cancer, movement disorders, neuroinfections, pain-related disorders, or acute brain injury.

[0117] In one exemplary embodiment, a capsid protein, nucleic acid, viral particle, and / or pharmaceutical composition may be used in combination with an immunosuppressant that mediates an immune response to AAV.

[0118] The composition may be administered simultaneously with, before or after, one or more other desired therapies for the treatment or prevention of neurodegenerative diseases, neuroinflammatory diseases, neurodevelopmental disorders, neuropsychiatric disorders, seizure-related disorders, cerebrovascular diseases, brain cancer, motor disorders, neuroinfections, pain-related disorders, or acute brain injury.

[0119] Diagnostic methods The capsid proteins, nucleic acids, or viral particles of the present invention can be used in diagnostic methods. In one exemplary embodiment, the capsid proteins, nucleic acids, or viral particles of the present invention can be used in diagnostic methods using positron emission tomography (PET) imaging. For example, the capsid proteins, nucleic acids, or viral particles of the present invention may be used in PET studies of the accumulation and clearance of systemic AAV capsids in the brain after parenteral administration.

[0120] How to target microglia The present invention also relates to a method for targeting cells using the AAV capsid protein of the present invention. In one embodiment, the present invention provides a method for targeting microglia using the AAV capsid protein of the present invention, comprising introducing a recombinant AAV vector encoding a target gene or silencing construct described herein, encapsulated within a capsid containing the capsid protein of the present invention, into a mammal.

[0121] kit The capsid proteins, nucleic acids, recombinant DNA, virus particles, and / or pharmaceutical compositions of the present invention can be packaged in a kit.

[0122] The present invention will be described below with reference to examples. [Examples]

[0123] material and method In vivo plasmid library construction AAV-CMVc-Cas9 was a gift from Juan Belmonte (Addgene plasmid #106431). pCAG-Cre-IRES2-GFP was a gift from Anjen Chenn (Addgene plasmid #26646). pUCmini-iCAP-PHP.eB was a gift from Viviana Gradinaru (Addgene plasmid #103005). pHelper plasmid was a gift from the Royal Cancer Research Foundation Viral Core Facility. The inventors PCR-amplified the appropriate sequences using the primers listed in Table 1. Using the AAV-CMVc-Cas9 plasmid backbone and AAV2 ITR sequence, fragments of the cap and rep genes from iCAP-PHP.eB, and the GFP sequence from Cre-IRES-GFP, the inventors cloned a novel selection library plasmid containing a GFP reporter using NEBBuilder HiFi DNA assembly. The inventors silenced the translations of VP1, VP2, and VP3 encoded in the PHP.eB capsid plasmid by inserting stop codons into each coding sequence. The silenced sequences were prepared and cloned using the NEBBuilder HiFi DNA Assembly as described above (Table 2). The silenced sequences were prepared by Integrated DNA Technologies and cloned as described above (Table 2). The random library was prepared by Sigma (Table 2).

[0124] All viral genomes were constructed using homology-based cloning methods with the DNA-HIFI Builder kit (NEB). The human Cd11b promoter was cloned from human DNA isolated from oral cells. DTA was cloned from genomic DNA of transgenic mice carrying the transgene. dsDNA of gRNA and shRNA sequences were prepared by oligo annealing of ssDNA oligos from Sigma-Agentur. The KASH domain was cloned from MEF cDNA. The GFP-targeting gRNA sequence was subcloned into a construct with a U6 promoter using BsaI-HFv2 (NEB) via Golden Gate cloning. The shRNA sequence was subcloned into an shRNA vector by restriction enzyme cloning. Modified capsid sequences for AAV2 and AAV6 were prepared by PCR. Primers were prepared to precisely sandwich the insertion sites of the newly identified sequences. Next, these sequences were added as extensions to the 5' end of each primer so that there was at least a 20 bp overlap between the inserted sequences. PCR was performed using Q5 polymerase to effectively generate linear dsDNA fragments of the vector sharing a 20 bp homologous sequence containing the newly added sequences at 5' and 3'. The PCR products were purified by column purification, and NEB DH5-α was transformed with 10 ng of purified DNA using a standard protocol. In all cases, positive clones were confirmed by Sanger sequencing.

[0125] Virus production protocols and in vivo transfection AAV production and purification were performed according to a previously reported protocol (Challis et al., 2018). Briefly, HEK293 cells were grown in 15 mm plastic dishes and triple-transfected with a transfer plasmid encoding pHelper plasmid, silenced PHP.eB capsid plasmid, and capsid library plasmid. After 5 days, the cells were lysed and the virus was precipitated using PEG. Viral particles were purified using OptiPrep density gradient medium (Sigma-America; D1556) and ultracentrifugation at 350,000 g. The viral layer was isolated and concentrated using an Amicon Ultra-15 centrifugal filter unit (Sigma-America; Z648043-24EA). AAV titer was determined using SYBR green qPCR. For in vivo administration of the above virus, 8-week-old mice were fixed and 5 × 10⁶ cells were administered. 11 The viral genome / virus was injected into the tail vein.

[0126] Immunofluorescence of tissue sections Mice were administered a lethal dose of pentobarbital and perfused transcardiacally with 4% paraformaldehyde (PFA) / PBS. The brains were excised and post-fixed with 4% PFA at room temperature for 2 hours. After rinsing with PBS, the tissue was incubated overnight in 20% sucrose solution (in PBS). The tissue was then embedded in OCT embedding medium (OCT-medium) (TissueTek) and stored at -80°C. 12 μm sections were obtained using a cryostat. The tissue sections were air-dried and stored at -80°C. Sections cut with a cryostat were dried at room temperature for 45 minutes. Slides were washed three times with PBS (5 minutes, room temperature) and blocked at room temperature for 1 hour with 0.3% PBST containing 10% NDS. The primary antibody was diluted with 0.1% PBST containing 5% NDS and incubated overnight at 4°C. The slides were washed three times with PBS for 10 minutes each. Next, a secondary antibody in blocking solution was applied at a concentration of 1:500 at room temperature for 2 hours. The slides were washed three times with PBS for 10 minutes each, with the first wash containing Hoechst33342 nuclear stain (2 μg / ml). Cover slips were mounted on the slides using FluoSave (CalBiochem). Image acquisition was performed using a Leica-SP5 microscope (Leica) and LAS software (Leica) or a Zeiss Observer A1 inverted microscope (Zeiss) and Zeiss Axivision software. Further image processing and analysis were performed using the ImageJ software package.

[0127] Isolation of single-cell suspension Adult male and female mice (8 weeks old) were decapitated after lethal injection with phenobarbital. The brains were rapidly removed and transferred to ice-cold isolation medium. The telencephalon and cerebellum were dissected in the isolation medium; the meninges and olfactory bulb were mechanically removed, and the brain tissue was mechanically cut into 1 mm sections. 3The tissue was divided into sections. These tissue sections were spun down at 100g for 1 minute at room temperature, and the tissue was washed with HBSS-(without Mg2+ and Ca2+, GIBCO). Each brain was then dispersed in 5 ml of isolation medium (homemade low-fluorescence Hibernate-A (Brainbits)) containing 34 U / ml papain (Worthington) and 20 μg / ml deoxyribonuclease IV (GIBCO). The mixture was then dispersed on a shaker (50 rpm) at 35°C for 30 minutes. Digestion was stopped by adding ice-cold HBSS-. The tissue was centrifuged (200 g, 3 minutes, room temperature), the supernatant was completely aspirated, and the tissue was resuspended in isolation medium (disintegration solution) supplemented with 2% B27 and 2 mM sodium pyruvate. The tissue was left in this solution for 5 minutes. To obtain a single-cell suspension, the tissue suspension was first pipetteed using a 5 ml serological pipette, and then heated over three flames. The tissue was crushed 10 times using a glass pipette (opening diameter > 0.5 mm). After each crushing step, the tissue suspension was allowed to settle (approximately 1-2 minutes), and the supernatant containing cells (approximately 2 ml) was transferred to a new tube. 2 ml of fresh crushing solution was added after each crushing. To remove any undigested tissue fragments that had been accidentally transferred, the collected supernatant was filtered through a 70 μm cell strainer and filtered with 90% isotonic Percoll (GE Healthcare, 17-0891-01, 10 × PBS). The mixture was transferred to a tube containing pH 7.2 (Lifetech). To the final volume, DMEM / F12 (Gibco), which is HEPES-containing and does not contain phenol red, was added and mixed to obtain a homogeneous suspension with a final Percoll concentration of 22.5%. The single-cell suspension was separated from the remaining debris particles by density gradient centrifugation (800 g, 20 minutes, room temperature, uninterrupted). The myelin debris and all cell-free layers were discarded, and the brain cell-containing phase (last 2 ml) and cell pellet were resuspended in HBSS+, combined in a new 15 ml tube, and centrifuged (300 g, 5 minutes, room temperature). The cell pellet was resuspended in erythrocyte lysis buffer (Sigma-Ace, R7757) and incubated at room temperature for 1 minute to remove erythrocytes. 10 ml of HBSS+ was added to this cell suspension and spun down (300 g, 5 minutes, room temperature).The cell pellet was resuspended in 0.5 ml of modified Milteny washing buffer (MWB, PBS at pH 7.3, containing 2 mM EDTA, 2 mM sodium pyruvate, and 0.5% BSA) supplemented with 10 ng / ml human recombinant insulin (Gibco).

[0128] Fluorescently labeled cell sorting Freshly isolated brain cells were stained with primary antibodies (anti-CD11b-PE and appropriate isotype controls) at 4°C for 15 minutes. The cells were washed and resuspended in FACS buffer. Cells were analyzed using Attune-NXT (Thermo Scientific) equipped with 405, 488, and 561 lasers. For correction, single staining of fluorophores was performed using beads (OneComp). The correction matrix was automatically calculated and applied using Attune software. Gating for quantification of Cd11b and GFP-positive cells was set based on appropriate FMO. A minimum of 50,000 cell singlets were recorded and used for quantification using FlowJo software (v10).

[0129] Culture of human microglia and mouse microglia Isolated microglia are seeded in 96-well plates at a density of 1 cell / well. The cells are incubated in DMEM F12 containing 60 μg / ml N-acetylcysteine ​​(Sigma-A), 10 μg / ml human recombinant insulin (Gibco), 1 mM sodium pyruvate (Gibco), 50 μg / ml apotransferrin (Sigma-A), 16.1 μg / ml putrescine (Sigma-A), 40 ng / ml sodium selenite (Sigma-A), and 20 ng / ml M-CSF. If cell viability is difficult, 330 ug / ml bovine serum albumin (Sigma-A) can be added, but this will reduce the cell proliferation potential. Microglia are incubated at 37°C, 5% CO2, and 5% O2.

[0130] After 10 days, replace 50% of the culture medium with medium containing 10 ng / ml M-CSF. Thereafter, the medium should be replaced every 3 days.

[0131] Next-generation sequencing for capsid sequence identification Cells were recovered by FACS and pelletized by centrifugation. The supernatant was removed to reduce the volume to approximately 20 μl, and 50 μl of Lucigen Rapid DNA Extraction Solution was added to all samples. These samples were then processed according to the manufacturer's instructions. Next, a sequencing library was prepared by two PCRs to add a unique barcode, a flow cell annealing barcode, and an Illumina index sequence. In the first PCR, 5 μl of DNA-containing Rapid Extraction Solution was used for a 50 μl PCR reaction. The PCR product was purified using 1.8 × AmpureXP beads and eluted into 40 μl. In the second PCR, 5% of this eluate was used as a template. Q5 polymerase (NEB) was used in all PCRs. The product was purified using AmpureXP beads (0.5 ×). Next, the library quality was evaluated by qPCR using a bioanalyzer. The pooled library was treated with 10% PhiX and sequenced using an Illumina MiSeq with an Illumina Nano (2×250bp) kit.

[0132] Example 1: PHP.eB cannot infect microglia in an in vivo environment. To confirm whether PHP.eB can infect microglial cells, the inventors prepared an AAV encoding spCas9 using a PHP.eB capsid and injected it into C57 / B6 mice. After 21 days, the mice were perfused and fixed, and brain tissue was stained with an anti-HA tagged antibody capable of detecting HA-tagged Cas-9 enzyme (Segel et al., 2019). To confirm that this was not related to the microglia's ability to reject Cas9 entry into the cells, the inventors repeated the experiment by injecting an AAV expressing green fluorescent protein (GFP) under the control of a CMV promoter using a PHP.eB capsid. Microglia were detected in the tissue by staining it with an Iba-1 antibody. Sections of the entire brain were examined for the presence of double-labeled GFP-expressing Iba-1-labeled cells. No double-labeled cells were found in the tissue (n=3) (Figure 2). These results were consistent with the original paper by Deverman et al. (2016), which presented data on the majority of cell populations within the CNS, excluding microglia.

[0133] Example 2: Creation of a random library to identify candidates for microglial infection. To investigate whether AAV can infect microglia via systemic circulation, the inventors constructed a novel random library of the virus for selection screening. To do this, the PHP.eB capsid plasmid (Figure 1A) was degraded using PCR, and individual target fragments were isolated. For the purpose of screening the random library, the target PHP.eB capsid sequence needed to be inserted into a novel transplasmid with a fluorescent label to confirm positive entry. The inventors extracted the backbone and ITR sequences from AAV-CMVc-Cas9 using restriction enzyme digestion (Segel et al., 2019). An expression cassette containing the p41 promoter and rep fragment t2a GFP, followed by a poly(A) tail start sequence, to drive the expression of the capsid library was constructed by PCR and assembled into a novel transfer plasmid by homology cloning using NEBuilder HIFI. To incorporate a random library sequence into the virus binding arm, the inventors amplified the capsid sequences of two sections, excluding the PHP.eB binding arm (amino acid sequence: TLAVPFK (starting at amino acid 589)), and replaced the 21 nucleotides encoding this sequence with the random sequence library (Figure 1B).

[0134] The PHP.eB capsid plasmid encoding the rep and cap sequences was used as the rep-cap plasmid for constructing the AAV. However, to eliminate capsid protein expression, the inventors inserted in-frame stop codons with reading frames for each capsid protein (VP1-3), as previously reported (Deverman et al., 2016) (Figure 1C). These stop codons do not alter the amino acid sequence of the assembly activation protein (AAP) expressed in a different reading frame within the Cap gene (Sonntag et al., 2010). These plasmids were used together with the pAAV helper plasmid to construct the novel AAV described herein.

[0135] Example 3: Microglia can be systemically infected using AAV technology. The virus library was placed in 150 μl of sterile PBS, 5 × 10⁶ 11 The viral genome was injected into C57 / B6 mice at the specified concentration. After 21 days, the mice were sacrificed, and the whole brain was removed for the purpose of preparing single-cell suspensions or perfused and fixed for immunohistochemical staining. Immunohistochemical staining for Iba-1 showed double-positive microglia indicating successful infection (Figures 3A and 3B). For the purpose of sorting positive GFP-labeled cells, fresh brains were homogenized into single-cell suspensions using an established method [Neumann et al., 2019] and labeled with CD11b / PE FACS antibody. Double-positive cells (CB11b + / GFP + The samples were sorted (Figure 3C), and DNA was extracted.

[0136] Example 4: Novel binding arms of AAV9 / PHP.eB enable entry into microglia. DNA obtained from GFP-positive microglia was isolated, and the viral genome region containing the random library sequence was amplified by PCR. The PCR product was subcloned using TOPO cloning. DNA obtained from individual bacterial colonies was extracted, and the region containing the random library sequence was sequenced using Sanger sequencing. Nine sequences were identified by TOPO cloning (Figures 4A-4D and Table 3). Of these, four sequences were used to construct novel capsid plasmids as described, and these were combined with GFP expression transfer plasmids to characterize each novel binding arm. Each of the four novel viruses was subjected to 5 × 10⁶ sampling. 11 The solution was injected into the tail vein of C57 / B6 mice at the specified concentration, and sorting was performed after 21 days. Immunohistochemical analysis of the tissue showed double-positive labeling of Iba-1-positive microglia containing GFP. To further confirm this finding, the inventors isolated microglia using CD11b beads via MACS, cultured them, and determined their viability (Figure 5). Finally, CD11b+ When the proportion of GFP-positive microglia was examined by FAC sorting of microglia, it varied between 45% and 81% when using four novel viruses (Figures 4A to 4D).

[0137] Example 5: Virus production Microglia-specific viruses were generated by using the CD11b promoter in conjunction with an mCherry fluorescent tag localized to the perinuclear lamina. While the inventors acknowledge that CD11b was proposed as a whole macrophage marker, their single-cell sequencing data demonstrates that, in the absence of acute brain injury, CD11b is an excellent marker for isolating microglia from brain parenchyma (Young et al., 2021). Three specific microglia viruses were generated along with appropriate controls, and 5 × 10⁶ microglia viruses were produced. 11 The drugs were injected into the tail vein of C57 / B6 mice at the specified concentrations and left for 4 weeks. Specifically, the entry of one brain and microglia of a novel capsid sequence containing AAV9, PHP.eB, and the insert sequence of the present invention (HGTAASH) was compared. Success of infection was determined by confirmation of transgene expression in cells labeled with the microglial marker Iba1 by IHC. When AAV9 was injected, no uptake into the brain parenchyma was shown at the concentrations and imaging settings used (Figure 6a). When PHP.eB was injected, uptake was shown in 42% of non-microglial brain cells, but no transgene expression was shown in microglia (Figure 6b). The novel microglial capsid entered 17% of non-microglial brain cells and 75% of microglia (Figure 6C). Immunohistochemical staining of brain sections confirmed the invasion of novel microglial capsids into microglia, which was not observed in the control samples AAV9 or PHP.eB (Figures 6d-6f).

[0138] Example 6: Upregulation of transgenes in microglia in vivo To evaluate whether measurable phenotypic effects can be obtained in microglia when a transgene is overexpressed, C57 / BL6 mice were subjected to 5 × 10¹⁶ transgenes encoding a diphtheria toxin (DTA) transgene under the control of the human CD11b promoter. 11 A number of virus particles were injected. When DTA is expressed in cells, it becomes toxic, resulting in cell death. Therefore, microglia expressing Cd11b, unlike other brain cell types, undergo apoptosis, leading to depletion of microglia in the brain. Flow cytometry revealed a significant decrease in microglia in the sample after DTA transgene expression compared to the control. Specifically, the number of microglia decreased from 3.2% to 0.24% as measured by flow cytometry (Figure 7a). Immunohistochemistry revealed a large tissue area of ​​microglia depletion (Figure 7b), and even more interestingly, a large number of Iba1-positive cells undergoing active apoptosis were identified (Figure 7c).

[0139] Example 7: Downregulation of gene expression in microglia in vivo The inventors then investigated whether the newly created viral capsids enabled infection of microglia by constructs that inhibit gene expression in host microglia. B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9 * 5×10 mice encoding shRNA for GFP, driven by the U6 promoter and CD11b promoter which regulate mCherry-KASH domain fusion protein expression. 11Cells were injected with 100% virus particles. Infection-positive cells expressed mCherry around the nuclear lamina, and cells that successfully expressed shRNA targeting GFP (expressed by all cells in CAG-Cas9-EGFP mice) should have reduced or absent GFP expression in the microglia compared to microglia obtained from mice infected with a control virus, a scramble control in which the shRNA does not target any genes (Figure 8a). Flow cytometry confirmed 75% microglial infection, as described above (see Figure 6; Example 5). Furthermore, in mice injected with shRNA targeting GFP, 58% of infected microglia showed a decrease in GFP levels compared to mice injected with control shRNA (Figure 8b). Immunohistochemistry confirmed a decrease in GFP expression in mCherry-positive microglia (Figure 8c).

[0140] Example 8: CRISPR gene editing of microglia To confirm whether the microglial genome can be edited using the CRISPR method, B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9 * ,-EGFP) mice are given 5×10⁶ mice encoding gRNA for GFP. 11 A number of virus particles were injected. To modulate infection, the virus was given a second expression cassette encoding an mCherry-KASH domain fusion protein under the control of the human Cd11b promoter. The GFP gRNA specifically targets the GFP gene and, as a result, downregulates GFP in any infected cell in this experiment. A microglial infection rate of 75% was confirmed by flow cytometry (Figure 9a). In mice injected with the virus encoding the GFP-targeting gRNA, 17% of infected microglia showed a decrease in GFP levels (Figure 9b). Immunohistochemistry confirmed a decrease in GFP expression in mCherry-positive microglia (Figure 9c).

[0141] Example 9: Infection of human microglia using a novel sequence To determine whether the novel virus could infect human cells, adult microglia were isolated from surgical biopsies. Cells were sorted using magnetic labeling with CD11b, resulting in 1 × 10⁶ cells. 10 Cells were infected with individual virus particles. The cells were cultured for 5 days, and mCherry expression and Iba1 expression were analyzed. In cells infected with the mCherry-encoding virus under the control of the human Cd11b promoter, mCherry expression was observed in 22% of all Iba1 cells after only 5 days of culture. Conversely, microglia infected with the AAV9 serotype or PhP.EBeB serotype did not show significant transgene expression in Iba1-positive cells (Figure 10). In summary, these cells reveal the infection efficiency in human microglia.

[0142] Example 10: Novel sequence of microglial infection To comprehensively and unbiasedly identify binding arm sequences capable of infecting microglia, sequences were identified by DNA sequencing. As described above, C57 / B6 mice were injected with a virus containing a random screening library of novel binding arms following the 588th amino acid of the VP1 capsid sequence. After 4 weeks of incubation, GFP-positive microglia were extracted using FACS. DNA from cells infected with the GFP transgene was prepared using Lucigen's rapid DNA extraction solution. A sequencing library was constructed by PCR, and sequences were identified using Illumina sequencing. A list of candidate amino acids for capsid sequence insertions that enable microglial infection is given in Table 4. The insertion sequences shown in Table 4 can be inserted into the AAV capsid protein in the same manner as described herein for any one of sequence numbers 1-9 or any of their variants.

[0143] Example 11: Demonstration of further insertion of binding arms into associated adenoviruses Finally, to investigate whether the insertion of the identified seven amino acid sequences alone was sufficient to enable infection, the same amino acid sequences were inserted into the capsid sequences of the AAV2 and AAV6 serotypes (Figure 11). The insertion sites were identified by pairwise structural alignment of the capsid sequences. To investigate whether the inserted sequences generally enable microglial infection, these sequences were inserted into homologous regions of the VP1 capsid sequences of AAV2 and AAV6. The inserted sequences were inserted after serine at position 487 in AAV6-VP1 and after glycine at position 586 in AAV2-VP1. Viruses encoding the mCherry reporter under the control of the human Cd11b promoter, based on these modified capsid proteins, were added to cultured mouse microglia. After infection, 34% (AAV6-HGTAASH) and 48% (AAV2-HGTAASH) of Iba1-positive cells expressed mCherry when infected with the modified capsid sequence. However, when the PHP.eB capsid sequence (control) was used, this reported low transduction efficiency dropped to 0%. Furthermore, testing with wild-type AAV2 capsid virus and wild-type AAV6 capsid virus did not show infection.

[0144] Unofficial sequence list Sequence ID 1 YAFGGEG Sequence ID 2 ALAVPFR Sequence ID 3 HGTAASH Sequence ID 4 IFVMLVR Sequence ID 5 LHQIPDS Sequence ID 6 LYPLIDL Sequence ID 7 AGTTGFP Sequence ID 8 RLAEITG Sequence ID 9 ALAVPFK Sequence ID 10 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPD PQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFP ADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDN VDADKVMITNEEEIKTTNPVATESYGQVATNHQSDGAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL Sequence ID 11 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDP QPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADV FMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSDGYAFGGEGAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL Sequence ID 12 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDP QPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADV FMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSDGALAVPFRAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL Sequence ID 13 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDP QPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADV FMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSDGHGTAASHAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL Sequence ID 14 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDP QPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADV FMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSDGIFVMLVRAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL Sequence ID 15 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDP QPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADV FMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSDGLHQIPDSAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL Sequence ID 16 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDP QPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADV FMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSDGLYPLIDLAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL Sequence ID 17 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDP QPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADV FMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSDGAGTTGFPAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL Sequence ID 18 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDP QPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADV FMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSDGRLAEITGAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL Sequence ID 19 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDP QPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADV FMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSDGALAVPFKAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL Sequence ID 20 Sequence ID 21 Sequence ID 22 Sequence ID 23 Sequence ID 24 Sequence ID 25 Sequence ID 26 Sequence ID 27 Sequence ID 28 Sequence ID 29 TATGCGTTTGGCGGAGAGGGG Sequence ID 30 GCTTTGGCGGTGCCTTTCAGG Sequence ID 31 CATGGGACAGCGGCATCGCAT Sequence ID 32 ATTTTTGTGATGCTCGTCAGG Sequence ID 33 TTGCATCAAATTCCTGATAGT Sequence ID 34 TTGTATCCTTTGATTGATCTC Sequence ID 35 GCTGGCACGACAGGTTTCCCG Sequence ID 36 CGTTTGGCGGAGATCACGGGG Sequence ID 37 GCTTTGGCGGTGCCTTTTAAG Sequence ID 38 CTAGGGGTTCCTGCGGCCTCTAGAGCCACCATGTTCAAATTTGAACTGACTAAGCGGCTC Sequence ID 39 AGCGGTCGCAGAGGAGCG Sequence ID 40 TCCCGCTCCTCTGCGACCGCTATGGCTGCCGATGGTTATCT Sequence ID 41 CAGATTACGAGTCAGGTATCTGGTG Sequence ID 42 GATACCTGACTCGTAATCTGGAGGGCAGAGGAAGTCTGCTAACATGCGG Sequence ID 43 ACTAGGGGTTCCTGCGGCCGCACACAAAAAACCAACACACAGATCTAATGGCTCCGGGTAT Sequence ID 44 GTCGACTTGCTTGTTAATCAATAAACCG sequence number 45 CCCATCACTCTGGTGGTTTTGTG sequence number 46 TTGATGAATCCTGGACCTG sequence number 47 AGTTCAGCTTGTCCTTGTTG sequence number 48 GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCNNNNNNNNNNNNNNNNNNNNCCCATCACTCTGGTGGTTTGTGGCCACTTGTCCATAGGA sequence number 49 ATGGCTGCCGATGGTTGACTTCCAGATTAACTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTAGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGC AACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAAGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGC AACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAATGAAAGAGGCCTGTAGATGATCTCCTCAGGAACCGGACTCCTCCGGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAA AAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCCAACCAATCGGAGACCTCCCGCAGCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACAGTGGCAGACAATAACTGAGGTGCCGATGGATGGGTAGTTCC sequence number 50 TCCTATGGACAAGTGGCCCAAACCACCAGAGTGATGGGGCTTTGGCGGTGCCTTTTAAGGCACAGGCGCAGACCGGTTGGGTTCAAAACCAAGGAATAC sequence number 51 TCCTATGGACAAGTGGCCCAAACCACCAGAGTGATGGGGCTTTGGCGGTGCCTTTCAGGGCACAGGGCGCAGACCGGTTGGGTTCAAAACCAAGGAATAC sequence number 52 TCCTATGGACAAGTGGCCACAAACCACCAGAGTGATGGGGCATGGGACAGCGGCATCGCATGCACAGGCGCAGACCGGTTGGGTTCAAAACCAAGGAATAC sequence number 53 TCCTATGGACAAGTGGCCCAAACCACCAGAGTGATGGGTATGCGTTTGGCGGAGAGGGGCACAGGCGCAGACCGGTTGGGTTCAAAACCAAGGAATAC sequence number 362 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGNGNLGRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPD PQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPA DVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASN TALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL Sequence ID 363 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTN VDIEKVMITDEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL Sequence ID 364 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGNGNLGRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDP QPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVF MIPS VMITDEEEIKATNPVATERFGTVAVNLQSSHGTAASHSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL Sequence ID 365 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDP QPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFM VPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEK VMITDEEEIRTTNPVATEQYGSVSTNLQRGHGTAASHNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0145] List of tables

[0146] [Table 1]

[0147] [Table 2]

[0148] Table 3

[0149] Table 4-1

[0150] Table 4-2

[0151] Table 4-3

[0152] Table 4-4

[0153] Table 4-5

[0154] Table 4-6

[0155] Table 4-7

[0156] Table 4-8

[0157] Table 4-9

Claims

1. Adeno-associated virus (AAV) capsid protein modified by inserting the following amino acid sequence, which is capable of transducing microglia or brain macrophages: (a) The amino acid sequence of SEQ ID NO: 1, or a variant thereof having one amino acid substitution; (b) The amino acid sequence of Sequence ID No. 2, or a variant thereof having one amino acid substitution; (c) The amino acid sequence of SEQ ID NO: 3, or a variant thereof having one amino acid substitution; (d) The amino acid sequence of SEQ ID NO: 4, or a variant thereof having one amino acid substitution; (e) The amino acid sequence of SEQ ID NO: 5, or a variant thereof having one amino acid substitution; (f) The amino acid sequence of SEQ ID NO: 6, or a variant thereof having one amino acid substitution; (g) The amino acid sequence of SEQ ID NO: 7, or a variant thereof having one amino acid substitution; (h) The amino acid sequence of SEQ ID NO: 8, or a variant thereof having one amino acid substitution; or (i) The amino acid sequence of Sequence ID No.

9.

2. The AAV capsid protein according to claim 1, modified by inserting the following amino acid sequence: (a) Amino acid sequence of Sequence ID No. 1; (b) Amino acid sequence of Sequence ID No. 2; (c) Amino acid sequence of Sequence ID No. 3; (d) Amino acid sequence of SEQ ID NO: 4; (e) Amino acid sequence of Sequence ID No. 5; (f) Amino acid sequence of SEQ ID NO: 6; (g) Amino acid sequence of SEQ ID NO: 7; or (h) Amino acid sequence of SEQ ID NO:

8.

3. The AAV capsid protein according to claim 1 or 2, wherein the unmodified AAV capsid protein is wild-type AAV1 capsid protein, wild-type AAV2 capsid protein, wild-type AAV5 capsid protein, wild-type AAV7 capsid protein, wild-type AAV8 capsid protein, wild-type AAV9 capsid protein, wild-type AAVrh10 capsid protein, wild-type AAVretrograde capsid protein, or wild-type PHP.eB capsid protein.

4. The AAV capsid protein according to claim 1 or 2, wherein the amino acid sequence is inserted into a sequence having at least 90% sequence identity with SEQ ID NO:

10.

5. The AAV capsid protein according to claim 4, wherein the amino acid sequence is inserted between amino acids 588 and 589 of SEQ ID NO: 10, or at an equivalent position in a sequence having at least 90% sequence identity with respect to SEQ ID NO:

10.

6. (a) Having the amino acid sequence of SEQ ID NO: 11; (b) Having the amino acid sequence of SEQ ID NO: 12; (c) Having the amino acid sequence of SEQ ID NO: 13; (d) Having the amino acid sequence of SEQ ID NO: 14; (e) Having the amino acid sequence of SEQ ID NO: 15; (f) Having the amino acid sequence of SEQ ID NO: 16; (g) Having the amino acid sequence of SEQ ID NO: 17; (h) Having the amino acid sequence of SEQ ID NO: 18; or, (i) Having the amino acid sequence of SEQ ID NO: 19, AAV capsid protein according to any one of claims 1 to 5.

7. A nucleic acid encoding the AAV capsid protein according to any one of claims 1 to 6.

8. (a) Having the nucleotide sequence of Sequence ID No. 20; (b) Having the nucleotide sequence of Sequence ID No. 21; (c) Having the nucleotide sequence of Sequence ID No. 22; (d) Having the nucleotide sequence of Sequence ID No. 23; (e) Having the nucleotide sequence of SEQ ID NO: 24; (f) Having the nucleotide sequence of Sequence ID No. 25; (g) Having the nucleotide sequence of SEQ ID NO: 26; (h) Having the nucleotide sequence of Sequence ID No. 27; or, (i) Having the nucleotide sequence of Sequence ID No. 28, The nucleic acid according to claim 7.

9. A host cell comprising the nucleic acid described in claim 7 or claim 8.

10. A viral particle comprising the AAV capsid protein described in any one of claims 1 to 6.

11. The viral particle according to claim 10, further comprising a recombinant polynucleotide encoding the following: (a) Target gene; (b) Gene editing constructs; (c) Antibody or antigen-binding fragment; or (d) A construct for gene silencing.

12. (a) The target gene is TREM2, CCL4 / CCL3, CD22, or CSF1R; (b) The gene editing construct targets TREM2, CCL4 / CCL3, CD22, or CSF1R; (c) The antibody or antigen-binding fragment binds to TREM2, CCL4 / CCL3, CD22, or CSF1R; or, (d) The gene silencing construct downregulates the expression of TREM2, CCL4 / CCL3, CD22, or CSF1R. The virus particle according to claim 11.

13. The viral particle according to claim 11 or claim 12, wherein the recombinant polynucleotide encoding the target gene, the gene editing construct, the antibody or antigen-binding fragment, or the gene silencing construct further comprises a microglia-specific promoter or enhancer, or a macrophage-specific promoter or enhancer.

14. The virus particle according to claim 13, wherein the microglia-specific promoter or enhancer is derived from the following: (a) TMEM119; (b) CX3CR1; or, (c) P2Y12 (P2RY12).

15. The viral particle according to claim 13, wherein the macrophage-specific promoter or enhancer is derived from the following: (a) CD11b; (b) CD68; (c) CSF1R; or (d) F4 / 80.

16. A host cell that produces the viral particles described in any one of claims 10 to 15.

17. A pharmaceutical composition comprising a virus particle according to any one of claims 11 to 15 and one or more pharmaceutically acceptable excipients.

18. A therapeutic agent and / or diagnostic agent comprising a virus particle according to any one of claims 11 to 15.

19. A recombinant AAV vector for use in a method for targeting microglia or brain macrophages, wherein the method comprises introducing the recombinant AAV vector into a mammal, the recombinant AAV vector encoding a target gene, a gene editing construct, an antibody or antigen-binding fragment, or a gene silencing construct, and further encoding a capsid protein, the capsid protein being modified by inserting an amino acid sequence having one of the amino acid sequences of SEQ ID NOs: 1 to 9.

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