Novel aav capsids for targeting the nervous system and uses thereof

Abstract

New adeno-associated virus (AAV) capsids are provided, as well as recombinant AAV (rAAV) virions comprising the capsids and a vector genome containing a transgene. The rAAV virions comprising the new capsids show improved CNS-targeted infection efficiency and / or increased expression levels of the transgene. Methods for treating a disease comprising administering to a subject in need thereof a rAAV virion comprising the AAV capsid are also provided.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to PCT application No. PCT / CN2022 / 107543, filed on July 22, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to the field of gene therapy. In particular, the present invention relates to novel adeno-associated virus (AAV) capsids and recombinant AAV particles comprising said capsids. rAAV particles comprising novel capsids exhibit improved central nervous system (CNS) targeting transduction efficiency and / or increased transgene expression levels.

[0004] sequence list

[0005] This disclosure includes a sequence list that is part of this disclosure. Background Technology

[0006] The statements in this section are provided only as background information in relation to this disclosure and do not necessarily constitute prior art.

[0007] Adeno-associated virus (AAV) is a non-enveloped virus with a diameter of about 20 nm that infects mammals such as humans and primates. To date, a large number of AAV serotypes have been identified, and it is known that many AAV serotypes exhibit variable tropism when infecting different animal species or cell types (Hastie, E and RJ Samulski, Adeno-associated virus at 50: a golden anniversary of discovery, research, and genetherapy success--a personal perspective. Hum Gene Ther, 2015, 26(5): 257-65).

[0008] The AAV genome is a single-stranded DNA (ssDNA) of approximately 4.7 kb, with inverted terminal repeat (ITR) sequences of about 145 bases at each end. The ITR sequences form Watson-Crick base pairs to create a T-shaped hairpin structure containing cis elements necessary for the replication and packaging of the AAV genome. The AAV genome contains two open reading frames (ORFs) flanking the ITR sequences. One ORF (also known as the “rep gene”) encodes four Rep proteins (Rep78, Rep68, Rep52, and Rep40). The other ORF (also known as the “cap gene”) encodes three capsid proteins (VP1, VP2, and VP3) and an assembly activation protein (AAP). The Rep proteins possess helicase activity and are required not only to induce capsid formation but also to integrate the AAV genome into the host cell chromosome. The AAV capsid is formed by the assembly of 60 molecules of VP1, VP2, and VP3 in a 1:1:10 ratio to form an icosahedral AAV shell. VP1, VP2, and VP3 differ primarily in their N-terminal regions, with VP1 containing a unique phospholipase A2 (PLA2) domain. Since the PLA2 domain is present only in VP1, the N-terminal region of VP1 is also known as the VP1 unique region (VP1u). The PLA2 domain is known to be exposed to the external environment of AAV particles under acidic conditions, but it is normally located within the AAV particle. Therefore, it is considered essential for AAV to escape from the endosome and translocate to the nucleus after entering the cell. Assembly activating protein (AAP) is a protein essential for the formation of the AAV capsid. In nature, AAV replication depends on the presence of helper viruses (such as adenoviruses and herpesviruses). In the presence of helper viruses, the AAV genome replicates in the host cell to form a complete AAV particle containing the AAV genome. The AAV particle is then released from the host cell. In the absence of helper viruses, the AAV genome can be maintained as an episome, or less frequently integrated into the host chromosome and become latent (Li, C and RJ Samulski, Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet, 2020. 21(4): 255-272).

[0009] AAV can infect a wide variety of cells, including dividing cells and post-mitotic non-dividing cells such as blood cells, muscle cells, and nerve cells. Since AAV is not pathogenic to humans, it carries a low risk of adverse effects. Furthermore, AAV viral particles are physicochemically stable. Therefore, AAV has recently gained attention for its value as a delivery vector for transferring exogenous genes into human tissues to treat congenital genetic diseases (Deverman, BE et al., Gene therapy for neurological disorders: progress and prospects. Nat Rev Drug Discov, 2018.17(10): p.767.). For example, AAV9 carrying the functional SMN gene has been developed to treat spinal muscular atrophy (Novartis, Zolgensma). Based on the contained capsid, AAV serotypes differ in their tropism and can therefore be used to target different tissue and cell types for gene delivery. However, gene delivery via naturally occurring AAV serotypes is limited by dose-limiting safety constraints and its largely broad tissue tropism, which restricts the development of effective AAV-based gene therapies, particularly for neurological disorders.

[0010] To expand the AAV toolkit for neural delivery, AAV capsids have been engineered through rational design (Lee, EJ, C.M. Guenther and J. Suh, Adeno-Associated Virus (AAV) Vectors: Rational Design Strategies for Capsid Engineering. Curr Opin Biomed Eng, 2018.7: pp. 58-63) or directed evolution (Pekrun, K. et al., Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors. JCI Insight, 2019.4(22)). For example, to improve neurotropism, a peptide insertion strategy was used to identify AAV-PHP.B and AAV-PHP.eb, which are derived from AAV9, a natural serotype that can cross the blood-brain barrier to infect nerve cells (Ravindra Kumar, S. et al., Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. Nat Methods, 2020.17(5): 541-550).

[0011] Furthermore, for the treatment of various neurological disorders, AAV serotypes with disease-related neuron-specific tissue tropism would be more desirable for both safety and efficacy considerations. For example, AAVs with frontal and temporal cortical tropism would be a preferred delivery tool for treating frontotemporal dementia. Other potential strategies may involve using AAVs targeting the dopaminergic systems in the striatum and midbrain to treat Parkinson's disease, using AAVs targeting motor neurons to treat spinal muscular atrophy (SMA) or amyotrophic lateral sclerosis (ALS), and using AAVs targeting dorsal root ganglia to relieve pain.

[0012] Therefore, there remains an unmet need for new AAV serotypes with more differentiated tissue tropism (such as improved CNS tropism). Summary of the Invention

[0013] The inventors of this invention designed and produced an AAV capsid library from which a series of novel capsids with improved CNS tropism have been identified. Each Cap gene in this library encodes a variant CAP polypeptide having an insertion of a random 7-amino acid (7-amino acid heptameric) extension at a site between amino acid positions 588 and 589 of the AAV9 capsid polypeptide (see [link to documentation]). Figure 1 ).

[0014] In a first aspect, the present invention relates to engineered capsid peptides of AAV, wherein the engineered capsid peptide is characterized by:

[0015] (1) It can be assembled into AAV particles.

[0016] (2) An insertion of seven amino acids at positions corresponding to amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein, wherein the amino acid numbering is based on the amino acid sequence as shown in SEQ ID NO:121, and

[0017] (3) Compared with wild-type capsid peptides of AAV9, it showed improved CNS tropism.

[0018] In a second aspect, the present invention relates to an rAAV particle comprising (a) an AAV capsid polypeptide of the first aspect and (2) a vector genome containing a transgene.

[0019] In a third aspect, the present invention relates to nucleic acids encoding the AAV capsid polypeptide of the first aspect, and AAV particles containing the AAV capsid.

[0020] In the fourth aspect, this application relates to recombinant vectors containing nucleic acids from the third aspect.

[0021] In a fifth aspect, this application relates to host cells containing the nucleic acid of the third aspect or the recombinant vector of the fourth aspect.

[0022] In a sixth aspect, the present invention relates to a pharmaceutical composition comprising (1) an AAV capsid and a vector genome of the first aspect; or rAAV particles of the second aspect; and (2) a pharmaceutically acceptable vector.

[0023] In a seventh aspect, the present invention relates to the use of the AAV capsid polypeptide, the nucleic acid, the rAAV particles, or the pharmaceutical composition in the manufacture of a medicament for delivering the transgene to a subject. In a more specific embodiment, the medicament is delivered to cells in the central nervous system, such as cortical neurons, motor neurons, dopaminergic neurons, astrocytes, or oligodendrocytes.

[0024] In an eighth aspect, the present invention relates to a method for treating a disease, the method comprising administering to a subject in need a therapeutically effective amount of rAAV particles of the second aspect or a pharmaceutical composition of the sixth aspect. Attached Figure Description

[0025] The following is a description of the accompanying drawings, which are presented for the purpose of illustrating the exemplary embodiments disclosed herein and not for the purpose of limiting the exemplary embodiments disclosed herein.

[0026] Figure 1 The structure of the plasmid construct contained in the library (rAAV-CapLib-in-cis) is shown, in which the Cap gene is engineered to include a 21nt insertion, resulting in the insertion of 7 amino acids at the position between Q588 and A589 in the encoded capsid polypeptide.

[0027] Figure 2 A protocol for in vivo screening in cynomolgus monkeys to identify AAVs with better CNS tropism is shown.

[0028] Figure 3 The enrichment scores of AAV vectors with AAV9 capsids, PHP-eB capsids, and 10 engineered capsids of this application (candidates ViVec-N001, ViVec-N002, ViVec-N003, ViVec-N004, ViVec-N005, ViVec-N006, ViVec-N020, ViVec-N021, ViVec-N023, and ViVec-N033) are shown in different brain regions. The first column indicates the sequence of the inserted amino acids.

[0029] Figure 4 The structure of the construct pAAV-CMV-GFP-lib, which is to be included in the main plasmid used to prepare the rAAV GFP-barcode library, is shown.

[0030] Figures 5-18 The relative enrichment scores of engineered capsid peptides are shown in different CNS tissues.

[0031] Figure 19 The expression levels of GFP transgenes delivered via rAAV for each engineered capsid polypeptide are shown.

[0032] Figure 20 The structure of the construct pAAV-CMV-luc-T2A-GFP, which is to be included in the master plasmid for in vitro validation of the engineered capsid peptide, is shown.

[0033] Figures 21A-21BResults of in vitro validation of four AAV capsid candidates in cultured human dopaminergic neurons are shown. (AB) Day 25, MOI (multiple of infection) = 1E+4 or 1E+5; (CD) Day 45, MOI = 1E+4 or 1E+5.

[0034] Figure 22 Results of in vitro validation of four AAV capsid candidates in cultured human cortical neurons (45 days after differentiation) are shown, MOI = 1E+4 or 1E+5.

[0035] Figure 23 Results of in vitro validation of four AAV capsid candidates in cultured human motor neurons (21 days post-differentiation) are shown, MOI = 1E+4 or 1E+5.

[0036] Figures 24A-24C Results of in vivo validation of two AAV capsid candidates (ViVec-N001 and ViVec-N004) in cynomolgus monkeys are shown. The intensity of GFP in different brain regions (including (A) striatum, (B) hippocampus, and (C) medulla) of monkeys treated with the AAV capsid candidates is shown.

[0037] Figure 25 The biodistribution of ViVec-N002 in the brain and spinal cord of cynomolgus monkeys is shown 21 days after intracerebroventricular (ICV) injection. Relative DNA levels of luciferase in each region are shown as fold changes (normalized relative to the AAV9 treatment group).

[0038] Figure 26 The biodistribution of ViVec-N022 and ViVec-N033 in different brain regions and peripheral organs of cynomolgus monkeys is shown 21 days after intracerebroventricular injection. The relative DNA levels of luciferase in each region are shown as fold changes (normalized relative to the AAV9 treatment group), N=2.

[0039] Figure 27 The relative RNA levels of luciferase in different brain regions and peripheral organs of cynomolgus monkeys 21 days after intracerebroventricular injection of AAV9, ViVec-N022, and ViVec-N033 are shown. The relative RNA levels of luciferase in each region are shown as fold changes (normalized relative to the AAV9 treatment group), N=2.

[0040] Figure 28 The expression levels of GFP in the striatum of mice 7 days after intrastriatal injection of ViVec-N001, ViVec-N002 and AAV9 are shown, N=3.

[0041] Figure 29The relative RNA levels of luciferase in the striatum of mice 21 days after intrastriatal injection of ViVec-N002, ViVec-N022, ViVec-N033 and AAV9 are shown (normalized relative to the AAV9 treatment group), N=6.

[0042] Figure 30 The relative RNA levels of luciferase in the spinal cord and cortex of rats (N=8, 4 males and 4 females) 21 days after intrathecal injection of ViVec-N022, ViVec-N033 and AAV9 are shown (normalized relative to the AAV9 treatment group). Detailed Implementation

[0043] definition

[0044] “AAV” refers to adeno-associated virus. “rAAV” refers to recombinant adeno-associated virus. In the context of this application, unless otherwise stated, “rAAV vector,” “rAAV viral particle,” and “rAAV viral unit” have the same meaning and refer to viral particles containing a vector genome encapsulated in an rAAV capsid. The rAAV vector genome lacks the rep and cap genes found in the wild-type AAV genome; therefore, rAAV is a replication-defective entity.

[0045] The "capsid" is formed by assembling three isoforms of "capsid protein" and "capsid polypeptide," namely VP1, VP2, and VP3. "Capsid protein" or "capsid polypeptide" does not typically indicate the type of isoform and should be understood to encompass all three isoforms.

[0046] The term "engineered capsid polypeptide" refers to an artificially modified capsid polypeptide that differs from the wild-type AAV capsid polypeptide by at least one amino acid. In a specific embodiment, the engineered capsid polypeptide of this application has a 7-mer insertion at a position corresponding to the location between Q588 and A589 of the wild-type AAV9 capsid protein (VP1) as shown in SEQ ID NO:121. In a preferred embodiment, the engineered capsid polypeptide of this application exhibits higher tissue tropism in one or more CNS tissues compared to the wild-type AAV9 capsid polypeptide. The amino acid numbering and insertion position are based on wild-type AAV9 VP1, as VP1 is the longest isoform of the three capsid proteins. Both VP2 and VP3 are N-terminal truncated forms of VP1, while still containing the region corresponding to the insertion position between Q588 and A589 of the AAV9 VP1 protein. It should be understood that, in the cases of VP2 (SEQ ID NO:123; its nucleotide sequence is shown in SEQ ID NO:128) and VP3 (SEQ ID NO:124; its nucleotide sequence is shown in SEQ ID NO:129), the insertion position number should be adjusted accordingly (between Q451 and A452 in VP2 and between Q386 and A387 in VP3).

[0047] The term "gene" refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding nucleotide sequence that participates in encoding RNA transcripts. The phrase "target gene" or its abbreviation "GOI" as used herein refers to the gene to be delivered via rAAV. In some embodiments, the GOI contains only the coding sequence of a polypeptide. In other embodiments, in addition to the coding sequence, the GOI contains non-coding regions and regulatory regions. In some embodiments, the GOI does not encode a polypeptide.

[0048] As used herein, the term "nucleotide" refers to the base-sugar-phosphate unit that makes up a nucleic acid sequence (e.g., a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence).

[0049] The terms “peptide,” “polypeptide,” or “protein,” which may be used interchangeably in this article, refer to a polymer of at least two amino acid residues linked by one or more peptide bonds.

[0050] When used herein with respect to polypeptides, particularly capsid polypeptides, the term "variant" refers to a polypeptide that differs from a wild-type polypeptide in one or more amino acid variations, but is structurally, activity, and / or functionally related to the wild-type polypeptide. The amino acid variations may be selected from one or more of insertions, deletions, substitutions, truncations, and modifications.

[0051] The term "AAV9" refers to wild-type AAV serotype 9.

[0052] "Dual plasmid system" refers to an AAV virus packaging system that uses two plasmids for co-transfection, one plasmid encoding the transgene and the other plasmid encoding the Rep gene, Cap gene, and Ad helper gene.

[0053] As used herein, the term "subject" refers to an individual, preferably a vertebrate, more preferably a non-human mammal, or a human. A non-human mammal may be a rodent (such as a mouse) or a non-human primate (such as an ape). The term "subject" may also encompass cells, tissues, and progeny of biological entities obtained in vivo or in vitro.

[0054] The phrase "effective amount" or "therapeutic effective amount" refers to an amount of composition (e.g., a composition containing rAAV viral particles) sufficient to produce the desired activity when delivered to a subject in need. The desired activity may encompass delaying the manifestation of the disorder, preventing or delaying the progression of the disorder, or alleviating at least one symptom of the disorder.

[0055] Methods for engineering capsid peptides

[0056] The method for preparing the engineered capsid peptide of this application is as follows: Figure 2 This is briefly illustrated below. In short, it involves constructing a DNA library containing a multinucleotide sequence encoding a single variant capsid polypeptide, each variant capsid polypeptide having a unique 7-mer insert at the desired position on the AAV9 capsid polypeptide. The types of 7-mer inserts included in the library should theoretically cover all possible amino acid combinations, approximately 20^7 in number. Furthermore, plasmid libraries can be constructed based on the DNA library of the capsid-coding sequence and used to express the variant capsid polypeptide in host cells to form AAV viral particles. The resulting AAV viral particle library can be delivered to animals (e.g., monkeys) for tissue tropism screening.

[0057] A "DNA library" refers to a library of variant polynucleotide sequences encoding variant capsid polypeptides. Specifically, the variant polynucleotide sequence contains a wild-type AAV9 capsid polypeptide sequence with seven NNK codon insertions corresponding to heptammonimeric amino acid insertions. The NNK codons can encode one of 20 amino acids at the insertion site. This method is known as "site saturation mutagenesis (SSM)," a powerful tool in protein engineering. The "NNK saturation mutagenesis strategy" is one of the most popular SSM strategies utilizing NNK degenerate codons. The chance of an NNK codon encoding a stop codon is very small.

[0058] "Plasmid library" refers to the collection of plasmid DNA libraries in this application.

[0059] "AAV library" or "AAV virus particle library" refers to a collection of AAV virus particles formed from capsids expressed by the plasmid library of this application in a host cell line. For example, the host cell line could be HEK293T cells.

[0060] A “tissue library” refers to a collection of AAV viral particles containing AAV sequences derived from RNA collected from certain tissues of test animals injected with the AAV library. Specifically, reverse transcription is performed to produce total cDNA from RNA extracted from the tissues, and viral DNA is amplified from the total DNA to produce a tissue-specific AAV library.

[0061] The "tropism" of rAAV for a specific tissue refers to the ability of a given rAAV to preferentially infect a given type of tissue or cell. Improved tropism indicates an increased ability to target the desired tissue, with a corresponding increase in infection of the target tissue.

[0062] As used herein, “CNS tropism” includes the tropism of rAAV for one or more CNS cells, CNS tissues, or brain regions. CNS tropism can be indicated by high transduction efficiency in one or more of the following tissues: parietal lobe, frontal lobe, insula, occipital lobe, temporal lobe, hippocampus, striatum, pons, medulla oblongata, midbrain, cerebellum, thalamus, hypothalamus, corpus callosum, pituitary gland, optic nerve, and olfactory bulb.

[0063] Engineered capsid peptides and the polynucleotides encoding them

[0064] In one aspect, this application provides a series of variant AAV capsid peptides. Compared to rAAV particles formed from the capsid of wild-type AAV9, rAAV particles formed from the variant AAV capsid peptides of this application exhibit increased tropism for one or more tissues or cells of the central nervous system.

[0065] Compared to wild-type AAV9, the variant AAV capsid polypeptide of this application exhibits improved CNS tissue tropism, comprising a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121. In some embodiments, the variant AAV capsid polypeptide of this application comprises a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, selected from the amino acid sequences shown in SEQ ID NO:1-60.

[0066] On the other hand, this application provides a nucleic acid molecule comprising a nucleotide sequence encoding a CNS-loving variant AAV capsid polypeptide of this application. In some embodiments, the nucleic acid molecule comprises a 21-nucleotide fragment, as shown in any one of SEQ ID NO: 61-120, inserted at a position between the codons Q588 and A589 of the wild-type AAV9 VP1 capsid protein, as shown in SEQ ID NO: 121.

[0067] AAV Library

[0068] On the other hand, this application provides an AAV library containing multiple variant AAV capsid peptides, each of which has a 7-amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, wherein each of the inserted 7 amino acids can be randomly selected from any of 20 amino acids. The AAV in the AAV library has at least 1 × 10⁻⁶ amino acids. 4 Species, at least 1×10 5 Species, at least 1×10 6 Species, at least 1×10 7 Species, at least 1×10 8 species or at least 1×10 9 A variety of variant capsid polypeptides.

[0069] On the other hand, this application provides a DNA or plasmid library containing nucleotide sequences encoding a plurality of variant AAV capsid polypeptides, each of the AAV capsid polypeptides having a 7-amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121. The DNA or plasmid library can express at least 1 × 10⁻⁶ nucleotides. 4 Species, at least 1×10 5 Species, at least 1×10 6 Species, at least 1×10 7 Species, at least 1×10 8 species or at least 1×10 9 A variety of variant capsid polypeptides. In a specific implementation, a nucleotide sequence in a DNA or plasmid library is generated by inserting seven NNK codons between the Q588 and A589 codons in the wild-type AAV9 VP1 capsid protein coding sequence using an NNK saturation mutagenesis strategy.

[0070] As described above, DNA libraries, plasmid libraries, and AAV libraries can be used to screen for novel variants of AAV capsid peptides or coding sequences that possess desired characteristics (such as expression efficiency, tissue tropism, etc.).

[0071] rAAV virus particles

[0072] In one aspect, this application provides rAAV viral particles comprising a capsid and a vector genome, wherein the capsid comprises the engineered capsid polypeptide of this application. The rAAV viral particles exhibit tropism in one or more CNS tissues. More preferably, compared to rAAV viral particles having a wild-type AAV9 capsid, the rAAV viral particles exhibit higher tropism and / or higher transduction efficiency in one or more CNS tissues.

[0073] For example, in one or more CNS tissues, rAAV viral particles containing the engineered capsid polypeptide of this application exhibit transduction efficiencies that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or 1000% higher than rAAV viral particles having a wild-type AAV9 capsid. For example, the transduction efficiency of rAAV in the target tissue can be measured and calculated based on the expression level of GOI delivered by AAV. The expression level of GOI can be detected by any conventional means in the art.

[0074] For example, the variant AAV capsid polypeptide of this application exhibits tissue tropism in one or more brain regions or CNS tissues selected from the parietal, frontal, temporal, occipital, insula, striatum, hippocampus, midbrain, thalamus, hypothalamus, cerebellum, medulla oblongata, pons, spinal cord, optic nerve, cervical dorsal root ganglion, and thoracic dorsal root ganglion. Preferably, rAAV with the variant AAV capsid of this application can exhibit improved tropism in one or more of the aforementioned brain regions or CNS tissues compared to rAAV with a wild-type capsid of AAV9. rAAV with the variant AAV capsid of this application can lead to desired distribution and / or expression of GOI in one or more brain regions or CNS tissues selected from the parietal, frontal, temporal, occipital, insula, striatum, hippocampus, midbrain, thalamus, hypothalamus, cerebellum, medulla oblongata, pons, spinal cord, optic nerve, cervical dorsal root ganglion, and thoracic dorsal root ganglion. Preferably, compared with rAAV having a wild-type capsid with AAV9, rAAV having the variant AAV capsid of this application can lead to increased expression of GOI in one or more of the brain regions or CNS tissues.

[0075] In specific embodiments, the rAAV viral particles of this application comprise a variant AAV capsid having an amino acid sequence obtained by introducing a 7-amino acid insertion, as shown in any one of SEQ ID NO:1-60, into the position between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein, as shown in SEQ ID NO:121. In a preferred embodiment, the 7-amino acid insertion is selected from any one of SEQ ID NO:1, 2, 4, 22, and 33. In a more preferred embodiment, the 7-amino acid insertion is selected from any one of SEQ ID NO:2, 22, and 33. In the context of this application, rAAVs having capsids comprising the 7-amino acid insertions of SEQ ID NO:1-60 are designated as ViVec-N001 to ViVec-N060, respectively.

[0076] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the apical lobe. Preferably, the rAAV exhibiting tropism for the apical leaf comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:30, SEQ ID NO:5, SEQ ID NO:22, SEQ ID NO:29, SEQ ID NO:2, SEQ ID NO:33, SEQ ID NO:26, SEQ ID NO:43, SEQ ID NO:7, SEQ ID NO:24, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:38, SEQ ID NO:04, SEQ ID NO:3, SEQ ID NO:23, SEQ ID NO:9, SEQ ID NO:40, SEQ ID NO:55, SEQ ID NO:27, SEQ ID NO:54, SEQ ID NO:49, SEQ ID NO:10, SEQ ID NO:44, SEQ ID NO:58, SEQ ID NO:121. SEQ ID NO:1, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:21, SEQ ID NO:16, SEQ ID NO:28, SEQ ID NO:20, and SEQ ID NO:45. Specifically, rAAV exhibits improved tropism for the apical lobe compared to wild-type rAAV with AAV9 or AAV-PHP.eB. More preferably, for rAAV containing a variant AAV capsid polypeptide and exhibiting tropism for the apical lobe, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any of the following: SEQ ID NO:30, SEQ ID NO:5, SEQ ID NO:22, SEQ ID NO:29, SEQ ID NO:2, SEQ ID NO:33, SEQ ID NO:26, SEQ ID NO:43, SEQ ID NO:7, and SEQ ID NO:24. This application also relates to the use of rAAV as described in this paragraph for delivering GOI to the apical lobe.

[0077] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the frontal lobe. Preferably, the rAAV exhibiting tropism for the frontal lobe comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:30, SEQ ID NO:29, SEQ ID NO:5, SEQ ID NO:33, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:15, SEQ ID NO:40, SEQ ID NO:38, SEQ ID NO:10, SEQ ID NO:54, SEQ ID NO:25, SEQ ID NO:47, SEQ ID NO:9, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:27, SEQ ID NO:121. SEQ ID NO:1, SEQ ID NO:44, and SEQ ID NO:39. Specifically, rAAV exhibits improved tropism for the frontal lobe compared to wild-type rAAVs having AAV9 or AAV-PHP.eB. More preferably, for rAAVs containing a variant AAV capsid polypeptide and exhibiting tropism for the frontal lobe, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any of the following: SEQ ID NO:30, SEQ ID NO:29, SEQ ID NO:5, SEQ ID NO:33, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:7, and SEQ ID NO:8. This application also relates to the use of rAAV as described in this paragraph for delivering GOI to the frontal lobe.

[0078] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the temporal lobe. Preferably, the rAAV exhibiting temporal lobe tropism comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:5, SEQ ID NO:33, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:40, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:15, SEQ ID NO:26, SEQ ID NO:51, SEQ ID NO:38, SEQ ID NO:3, SEQ ID NO:25, SEQ ID NO:35, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:47, SEQ ID NO:9, SEQ ID NO:49, SEQ ID NO:28, SEQ ID NO:121. SEQ ID NO:55, SEQ ID NO:20, SEQ ID NO:27, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:1, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO:11. Specifically, rAAV exhibits improved tropism for the temporal lobe compared to wild-type rAAVs having AAV9 or AAV-PHP.eB. More preferably, for rAAVs containing a variant AAV capsid polypeptide and exhibiting tropism for the temporal lobe, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any of the following: SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:5, SEQ ID NO:33, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:40, and SEQ ID NO:7. This application also relates to the use of rAAVs as described in this paragraph for the delivery of GOI to the temporal lobe.

[0079] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the occipital lobe. Preferably, the rAAV exhibiting tropism for the occipital lobe comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:22, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:33, SEQ ID NO:29, SEQ ID NO:7, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:40, SEQ ID NO:9, SEQ ID NO:38, SEQ ID NO:54, SEQ ID NO:23, SEQ ID NO:49, SEQ ID NO:10, SEQ ID NO:55, SEQ ID NO:47, SEQ ID NO:1, SEQ ID NO:27, SEQ ID NO:44, SEQ ID NO:1, SEQ ID NO:27, SEQ ID NO:44, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:49, SEQ ID NO:10, SEQ ID NO:55, SEQ ID NO:47, SEQ ID NO:1 ...49, SEQ ID NO:10, SEQ ID NO:55, SEQ ID NO:47, SEQ ID NO:1, SEQ ID NO:28, SEQ ID NO:49, SEQ ID NO:10, SEQ ID NO:55, SEQ ID NO:47, SEQ ID NO:1, SEQ ID NO:28, SEQ ID NO:58, SEQ ID NO:25, SEQ ID NO:28, and SEQ ID NO:39. Specifically, rAAV exhibits improved tropism for the occipital lobe compared to wild-type rAAV with AAV9 or AAV-PHP.eB. More preferably, for rAAV containing a variant AAV capsid polypeptide and exhibiting tropism for the occipital lobe, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any of the following: SEQ ID NO:22, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:33, SEQ ID NO:29, SEQ ID NO:7, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:8, and SEQ ID NO:24. This application also relates to the use of rAAV as described in this paragraph for delivering GOI to the occipital lobe.

[0080] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for insular leaves. Preferably, the rAAV exhibiting tropism for island leaves comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:30, SEQ ID NO:29, SEQ ID NO:40, SEQ ID NO:5, SEQ ID NO:23, SEQ ID NO:33, SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:15, SEQ ID NO:7, SEQ ID NO:24, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:38, SEQ ID NO:25, SEQ ID NO:51, SEQ ID NO:20, SEQ ID NO:35, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:47, SEQ ID NO:9, SEQ ID NO:28, SEQ ID NO:121 ... SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:43, SEQ ID NO:27, SEQ ID NO:55, SEQ ID NO:1, SEQ ID NO:50, SEQ ID NO:17, SEQ ID NO:39, SEQ ID NO:44, and SEQ ID NO:11. Specifically, rAAV exhibits improved tropism for insular leaves compared to wild-type rAAV with AAV9 or AAV-PHP.eB. More preferably, for rAAV containing a variant AAV capsid polypeptide and exhibiting tropism for insular leaves, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any of the following: SEQ ID NO:30, SEQ ID NO:29, SEQ ID NO:40, SEQ ID NO:5, SEQ ID NO:23, SEQ ID NO:33, SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:21, and SEQ ID NO:22. This application also relates to the use of rAAV, as described in this paragraph, for delivering GOI to the island leaf.

[0081] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting striatal tropism. Preferably, the rAAV exhibiting striatal tropism comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:30, SEQ ID NO:29, SEQ ID NO:5, SEQ ID NO:21, SEQ ID NO:43, SEQ ID NO:33, SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:23, SEQ ID NO:22, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:24, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:26, SEQ ID NO:40, SEQ ID NO:9, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:20, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:28, SEQ ID NO:121. SEQ ID NO:49, SEQ ID NO:41, SEQ ID NO:55, and SEQ ID NO:1. Specifically, rAAV midbrain exhibits improved striatal tropism compared to wild-type rAAVs with AAV9 or AAV-PHP.eB capsids. More preferably, for rAAVs containing a variant AAV capsid polypeptide and exhibiting striatal tropism, the variant AAV capsid polypeptide comprises a 7-amino acid insertion as shown in any of the following: SEQ ID NO:30, SEQ ID NO:29, SEQ ID NO:5, SEQ ID NO:21, SEQ ID NO:43, SEQ ID NO:33, SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:23, SEQ ID NO:22, and SEQ ID NO:1. This application also relates to the use of rAAV as described in this paragraph for delivering GOI to the striatum.

[0082] In one embodiment, this application provides rAAV containing a variant AAV capsid polypeptide and exhibiting tropism for the hippocampus. Preferably, the rAAV exhibiting tropism for the hippocampus comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:5, SEQ ID NO:30, SEQ ID NO:23, SEQ ID NO:33, SEQ ID NO:22, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:21, SEQ ID NO:15, SEQ ID NO:38, SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:44, SEQ ID NO:55, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:40, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:121 ... SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:1, SEQ ID NO:58, SEQ ID NO:20, SEQ ID NO:3, and SEQ ID NO:36. Specifically, rAAV exhibits improved tropism for the hippocampus compared to wild-type rAAVs having AAV9 or AAV-PHP.eB. More preferably, for rAAVs containing a variant AAV capsid polypeptide and exhibiting tropism for the hippocampus, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any of the following: SEQ ID NO:5, SEQ ID NO:30, SEQ ID NO:23, SEQ ID NO:33, SEQ ID NO:22, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, and SEQ ID NO:1. This application also relates to the use of rAAV as described in this paragraph for the delivery of GOI to the hippocampus.

[0083] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting midbrain tropism. Preferably, the rAAV exhibiting midbrain tropism comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:30, SEQ ID NO:5, SEQ ID NO:29, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:33, SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:15, SEQ ID NO:26, SEQ ID NO:37, SEQ ID NO:47, and SEQ ID NO:40. In particular, rAAV exhibits improved midbrain tropism compared to rAAV having a wild-type capsid with AAV9 or AAV-PHP.eB. More preferably, for rAAV containing a variant AAV capsid polypeptide and exhibiting midbrain tropism, the variant AAV capsid polypeptide comprises a 7-amino acid insertion as shown in any of the following: SEQ ID NO:30, SEQ ID NO:5, SEQ ID NO:29, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:33, SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:24. This application also relates to the use of rAAV as described in this paragraph for delivering GOI to the midbrain.

[0084] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the thalamus. Preferably, the rAAV exhibiting thalamic tropism comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:30, SEQ ID NO:29, SEQ ID NO:40, SEQ ID NO:5, SEQ ID NO:33, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:2, SEQ ID NO:23, SEQ ID NO:7, SEQ ID NO:26, SEQ ID NO:24, SEQ ID NO:15, SEQ ID NO:8, SEQ ID NO:3, SEQ ID NO:25, SEQ ID NO:54, SEQ ID NO:47, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:38, SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:49, SEQ ID NO:1 ...29, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:49, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:49, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:2 NO:37 and SEQ ID NO:51. Specifically, rAAV exhibits improved tropism for the thalamus compared to wild-type rAAVs having AAV9 or AAV-PHP.eB capsids. More preferably, for rAAVs containing a variant AAV capsid polypeptide and exhibiting tropism for the thalamus, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any of the following: SEQ ID NO:30, SEQ ID NO:29, SEQ ID NO:40, SEQ ID NO:5, SEQ ID NO:33, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:2, SEQ ID NO:23, and SEQ ID NO:7. This application also relates to the use of rAAV as described in this paragraph for the delivery of GOI to the thalamus.

[0085] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the hypothalamus. Preferably, the rAAV exhibiting hypothalamic tropism comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:30, SEQ ID NO:3, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:23, SEQ ID NO:33, SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:7, SEQ ID NO:24, SEQ ID NO:40, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:38, SEQ ID NO:15, SEQ ID NO:10, SEQ ID NO:25, SEQ ID NO:36, SEQ ID NO:47, SEQ ID NO:54, SEQ ID NO:53, SEQ ID NO:20, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:121 ... SEQ ID NO:35, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:28, SEQ ID NO:51, SEQ ID NO:12, SEQ ID NO:44, and SEQ ID NO:49. Specifically, rAAV exhibits improved hypothalamic tropism compared to wild-type rAAVs having AAV9 or AAV-PHP.eB. More preferably, for rAAVs containing a variant AAV capsid polypeptide and exhibiting hypothalamic tropism, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any of the following: SEQ ID NO:30, SEQ ID NO:3, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:23, SEQ ID NO:33, SEQ ID NO:2, and SEQ ID NO:22. This application also relates to the use of rAAV as described in this paragraph for the delivery of GOI to the hypothalamus.

[0086] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting cerebellar tropism. Preferably, the rAAV exhibiting cerebellar tropism comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:30, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:23, SEQ ID NO:2, SEQ ID NO:33, SEQ ID NO:22, SEQ ID NO:29, SEQ ID NO:7, SEQ ID NO:26, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:21, SEQ ID NO:40, SEQ ID NO:3, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:38, SEQ ID NO:25, SEQ ID NO:20, SEQ ID NO:47, SEQ ID NO:9, SEQ ID NO:49, SEQ ID NO:28, SEQ ID NO:121. SEQ ID NO:55, SEQ ID NO:1, and SEQ ID NO:44. Specifically, rAAV exhibits improved cerebellar tropism compared to wild-type rAAVs having AAV9 or AAV-PHP.eB capsids. More preferably, for rAAVs containing a variant AAV capsid polypeptide and exhibiting cerebellar tropism, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any of the following: SEQ ID NO:30, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:23, SEQ ID NO:2, SEQ ID NO:33, SEQ ID NO:22, SEQ ID NO:29, SEQ ID NO:7, and SEQ ID NO:26. This application also relates to the use of rAAVs as described in this paragraph for the delivery of GOI to the cerebellum.

[0087] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting medullophilicity. Preferably, the rAAV exhibiting medullary tropism comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:29, SEQ ID NO:5, SEQ ID NO:30, SEQ ID NO:23, SEQ ID NO:40, SEQ ID NO:33, SEQ ID NO:22, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:6, SEQ ID NO:2, SEQ ID NO:24, SEQ ID NO:8, SEQ ID NO:53, SEQ ID NO:15, SEQ ID NO:26, SEQ ID NO:3, SEQ ID NO:38, SEQ ID NO:28, SEQ ID NO:21, SEQ ID NO:47, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:25, SEQ ID NO:54, SEQ ID NO:121, SEQ ID NO:29, SEQ ID NO:5, SEQ ID NO:30, SEQ ID NO:23, SEQ ID NO:40, SEQ ID NO:33, SEQ ID NO:22, SEQ ID NO:4, SEQ ID NO:38, SEQ ID NO:28, SEQ ID NO:21, SEQ ID NO:47, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:25, SEQ ID NO:54, SEQ ID NO:121, SEQ ID NO:29 ... SEQ ID NO:49, SEQ ID NO:37, and SEQ ID NO:1. Specifically, rAAV exhibits improved medullary tropism compared to wild-type rAAVs having AAV9 or AAV-PHP.eB. More preferably, for rAAVs containing a variant AAV capsid polypeptide and exhibiting medullary tropism, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any of the following: SEQ ID NO:29, SEQ ID NO:5, SEQ ID NO:30, SEQ ID NO:23, SEQ ID NO:40, SEQ ID NO:33, SEQ ID NO:22, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:6, SEQ ID NO:2, SEQ ID NO:24, SEQ ID NO:8, SEQ ID NO:53, SEQ ID NO:15, SEQ ID NO:26, SEQ ID NO:3, and SEQ ID NO:1. This application also relates to the use of rAAV as described in this paragraph for delivering GOI to the medulla.

[0088] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the pons. Preferably, the rAAV exhibiting tropism for the pons comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:30, SEQ ID NO:5, SEQ ID NO:23, SEQ ID NO:22, SEQ ID NO:33, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:29, SEQ ID NO:7, SEQ ID NO:26, SEQ ID NO:3, SEQ ID NO:24, SEQ ID NO:15, SEQ ID NO:8, SEQ ID NO:40, SEQ ID NO:21, SEQ ID NO:38, SEQ ID NO:54, SEQ ID NO:20, SEQ ID NO:47, SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:28, SEQ ID NO:25, SEQ ID NO:6, SEQ ID NO:121, SEQ ID NO:29 ...20, SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:28, SEQ ID NO:25, SEQ ID NO:6, SEQ ID NO:121, SEQ SEQ ID NO:49, SEQ ID NO:41, and SEQ ID NO:37. Specifically, rAAV exhibits improved tropism for the pons compared to wild-type rAAVs having AAV9 or AAV-PHP.eB. More preferably, for rAAVs containing a variant AAV capsid polypeptide and exhibiting tropism for the pons, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any of the following: SEQ ID NO:30, SEQ ID NO:5, SEQ ID NO:23, SEQ ID NO:22, SEQ ID NO:33, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:29, SEQ ID NO:7, and SEQ ID NO:26. This application also relates to the use of rAAV as described in this paragraph for the delivery of GOI to the pons.

[0089] In one embodiment, this application provides rAAV containing a variant AAV capsid polypeptide and exhibiting tropism for the spinal cord. Preferably, the rAAV exhibiting tropism for the spinal cord comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein as shown in SEQ ID NO:121, as shown in any of the following: SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:40, SEQ ID NO:5, SEQ ID NO:30, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:38, SEQ ID NO:25, SEQ ID NO:21, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:28, SEQ ID NO:49, SEQ ID NO:47, SEQ ID NO:54, SEQ ID NO:121. SEQ ID NO:51, SEQ ID NO:20, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:53, and SEQ ID NO:37. Specifically, rAAV exhibits improved tropism for the spinal cord compared to wild-type rAAVs having AAV9 or AAV-PHP.eB. More preferably, for rAAVs containing a variant AAV capsid polypeptide and exhibiting tropism for the spinal cord, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any of the following: SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:40, SEQ ID NO:5, SEQ ID NO:30, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:7. This application also relates to the use of rAAVs as described in this paragraph for the delivery of GOI to the spinal cord. The spinal cord may be the cervical spinal cord and / or the thoracic spinal cord.

[0090] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for dopaminergic (DA) neurons. Preferably, the rAAV exhibiting tropism for DA neurons comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion as shown in any of the following: SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:46, SEQ ID NO:2, SEQ ID NO:28, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:49, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:8, SEQ ID NO:26, SEQ ID NO:15, SEQ ID NO:40, SEQ ID NO:38, SEQ ID NO:30, SEQ ID NO:29, SEQ ID NO:54, SEQ ID NO:3, SEQ ID NO:20, SEQ ID NO:47, SEQ ID NO:59, SEQ ID NO:43, SEQ ID NO:9, SEQ ID NO:57, SEQ ID NO:53, SEQ ID NO:1 ...20, SEQ ID NO:47, SEQ ID NO:59, SEQ ID NO:4 SEQ ID NO:16, SEQ ID NO:58, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:35, SEQ ID NO:55, and SEQ ID NO:17. Preferably, the variant AAV capsid polypeptide comprises a 7-amino acid insertion as shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:23, and rAAV having the variant AAV capsid polypeptide exhibits a preference for improving DA neurons compared to rAAV having a wild-type capsid with AAV9. More preferably, the variant AAV capsid polypeptide comprises a 7-amino acid insertion as shown in any one of SEQ ID NO:2 and SEQ ID NO:4, and rAAV having the variant AAV capsid polypeptide exhibits a preference for improving DA neurons compared to rAAV having a wild-type capsid with AAV-PHP.eB. This application also relates to the use of rAAV as described in this paragraph for delivering GOI to DA neurons.

[0091] In one embodiment, this application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for cortical neurons. Preferably, the rAAV exhibiting tropism for cortical neurons comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion as shown in any one of SEQ ID NO:1-60. Preferably, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any one of SEQ ID NO:1 and SEQ ID NO:2, and the rAAV having the variant AAV capsid polypeptide exhibits improved tropism for cortical neurons compared to rAAV having a wild-type capsid with AAV9. This application also relates to the use of the rAAV as described in this paragraph for delivering GOI to cortical neurons.

[0092] In one embodiment, this application provides an rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for motor neurons (e.g., immature or mature motor neurons, such as motor neurons derived from iPSCs). Preferably, the rAAV exhibiting tropism for motor neurons comprises a variant AAV capsid polypeptide containing a 7-amino acid insertion as shown in any one of SEQ ID NO:1-60. Preferably, the variant AAV capsid polypeptide contains a 7-amino acid insertion as shown in any one of SEQ ID NO:1 and SEQ ID NO:2 and the rAAV having the variant AAV capsid polypeptide exhibits improved tropism for motor neurons compared to rAAV having a wild-type capsid with AAV9. This application also relates to the use of the rAAV as described in this paragraph for delivering GOI to motor neurons.

[0093] use

[0094] The rAAV of this application is particularly suitable for delivering GOIs to CNS systems due to its improved CNS tropism. Therefore, it can be used for a variety of applications, including therapeutic uses for treating CNS diseases or for diagnostic purposes. For specific types of target tissues or cells in the CNS, variant AAV capsid peptides can be selected based on the sequencing and tissue preferences provided in this disclosure.

[0095] In an embodiment for therapeutic use, a therapeutically effective amount of rAAV viral particles is administered to the subject. The therapeutically effective amount can be from 1E+9vg to 1E+16vg, such as 1E+9vg, 1E+10vg, 1E+11vg, 1E+12vg, 1E+13vg, 1E+14vg, 1E+15vg, or 1E+16vg.

[0096] The rAAV of this application can be administered via various routes. For example, the rAAV of this application can be administered to the subject systemically or locally. For example, routes of administration include, but are not limited to, intravenous, intramuscular, intracerebellomedullary cistern injection, intrathecal injection, intravitreal injection, intraparenchymal injection, or intraventricular injection.

[0097] Example

[0098] Example 1. Preparation of AAV capsid library

[0099] A mutagenic AAV library containing a mutant Cap gene (VP1 gene, the coding sequence of the wild-type AAV9 capsid protein VP1 shown in SEQ ID NO:122) was generated using an NNK saturation mutagenesis strategy adapted from prior art (Ravindra Kumar, S. et al., ibid.) for a first round of screening (R1 library, or "R1 library"). The mutant capsid coding sequence contained in the R1 library has a random 21-base extension consisting of 7-NNK codons, thereby encoding a mutant capsid protein having a heptameric (7 amino acids or a heptameric) inserted at the position between amino acid position 588(Q) and amino acid position 589(A) of the wild-type AAV9 VP1 protein having the amino acid sequence shown in SEQ ID NO:121. As is generally understood in the art, N can be any of A, C, G, and T, and K can be G or T. Thus, the entire library can cover all possible combinations of 7 amino acids (see Figure 1 The first round of library construction is described in detail below. A brief description of the workflow is provided in [link to workflow description]. Figure 2 As shown in the image.

[0100] Synthesis of random fragments

[0101] First, short, random nucleotide fragments are generated for insertion into the coding sequence of the AAV9 VP1 capsid protein. Each fragment contains the coding sequence for a random heptamer. The random nucleotide fragments are synthesized by PCR using a degenerate primer set containing random 21 bases encoding the heptamer.

[0102] Skeletal digestion

[0103] The vector (rAAV-Cap-in-cis) was linearized using KpnI and AgeI (NEB), and the region encoding amino acids from position 546 (inclusive) to position 599 (inclusive) of the AAV9VP1 protein was removed. The enzymatic digestion mixture is shown below.

[0104] reagents quantity carrier 12ug KpnI 10uL AgeI 10uL Cutsmart 10uL <![CDATA[ddH2O]]> Up to 100uL

[0105] Digestion was performed overnight at 37°C, and the digested vector backbone was purified using the Zymoclean Gel DNA Recovery Kit (ZYMORESEARCH, D4008).

[0106] connect

[0107] At 50°C, random library fragments were placed in... The DNA was ligated into a linearized backbone in a HiFi DNA assembly premix (NEB, E2621) for 1 hour to produce the R1 library plasmid (rAAV-CapLib-in-cis).

[0108] plasmid transformation

[0109] After ligation, the mixture was diluted 4-fold, and 10 μL of the diluted ligation mixture was added to 100 μL of TransStbl3 chemocompetent cells (TransGen Biotech, CD521-02) and incubated on ice for 30 min, followed by heating at 42 °C for 30 s and then placing on ice for 3 min. Next, 1 mL of SOC medium was added to the cells and incubated on a shaker at 37 °C for 2 h. The cells were then transferred to 20 mL of LB medium containing 50 μg / mL ampicillin and incubated at 37 °C for 4 h, followed by transfer to 300 mL of LB medium containing 100 μg / mL ampicillin for further growth for 12 h, after which plasmids were extracted.

[0110] plasmid extraction

[0111] Following the manufacturer's instructions, plasmids for rAAV generation were extracted using the Purelink Hipure Filter Maxi Kit (Thermo, K210018). Briefly, 300 mL of overnight culture was centrifuged at 8,000 rpm to collect bacteria, and resuspended in 15 mL of R3, followed by 15 mL of L7 for lysis for 5 min. Then, 15 mL of N3 was added for 10 min and centrifuged at 12,000 rpm. The supernatant was then transferred to a column treated with EQ1. 40 mL of W8 was added to wash the column twice, followed by 15 mL of E4 to elute the plasmid DNA into a new centrifuge tube. 10.5 mL of isopropanol was added to the tube, and the mixture was centrifuged at 9,000 rpm for 1 h at 4°C to collect the plasmid DNA. The supernatant was removed, and the precipitated plasmid DNA was transferred to a 1.5 mL centrifuge tube, resuspended, and washed twice with 70% ethanol. The tube was then placed at 65°C to allow the ethanol to evaporate, and 200 μL of sterile water was added to dissolve the plasmid DNA.

[0112] Production and purification of rAAV

[0113] HEK293T cells were cultured in Dürbeco modified Eagle medium (DMEM, Gibco, C11995500BT) supplemented with 10% fetal bovine serum (FBS, Gibco, 10099141) and 1% penicillin-streptomycin (PS, Gibco, 15140-122).

[0114] For rAAV library generation, the rAAV-CapLib-in-cisDNA library and AAV helper plasmid were co-transfected into HEK293T cells in 15 cm culture dishes using lipofectamine LTX and plus reagent (Invitrogen, 94756). After incubation for 6–8 h, the medium was replaced with DMEM (5% FBS, 1% PS). The first harvest was performed 48 h post-transfection by collecting rAAV from the supernatant. A second harvest was performed 72 h post-transfection, with cells collected along with the culture medium.

[0115] For iodixanol purification, HEK293T cells from eight 15cm plates were lysed in 30mL of lysis buffer. 60% iodixanol (Axis-Shield, AS1114542) was diluted to 15%, 25%, 40%, and 60% in 10×PBS-MK (10×PBS, 10mM MgCl2·6H2O, 25mM KCl). NaCl was added to the 15% phase at a final concentration of 1M. To facilitate interface differentiation within the gradient, 2μL of phenol red was added to 1mL of 25% and 60% iodixanol solutions, respectively. 15mL of treated cell lysate, 9mL of 15% iodixanol solution, 6mL of 25% iodixanol solution, 5mL of 40% iodixanol solution, and 5mL of 60% iodixanol solution were added from top to bottom to 39mL Quick-Seal tubes (Beckman, 344326). After centrifugation at 60,000 rpm for 1.5 h in a 70Ti rotor at 18 °C, rAAV was collected from the 40% phase and treated with a solution containing 0.001% Pluronic acid. TM The rAAV was ultrafiltered with PBS using the F-68 nonionic surfactant (Gibco, 24040032) and concentrated using an Amicon Ultra-4 centrifugal filter (Millipore, UFC810096). The purified rAAV was stored at -80°C as an R1 rAAV library.

[0116] R1 library representation

[0117] Deep sequencing was performed on both the R1 DNA library rAAV-CapLib-in-cis and the final R1 rAAV library. 376,441 unique insertion sequences were identified in the R1 rAAV library prepared from the R1 DNA library.

[0118] Example 2. First round of in vivo screening

[0119] This embodiment describes the process of the first round of AAV capsid library screening, which generated more than 8,000 candidate variants of different capsid protein sequences.

[0120] Monkeys that have not been treated with AAV were screened using an in vitro neutralizing antibody assay.

[0121] AAV neutralizing antibodies (NAb) in laboratory animals were determined by in vitro neutralizing antibody assay to identify and select untreated monkeys for screening rAAV libraries. Serum was collected from 1-year-old cynomolgus macaques one month prior to injection (n = 3 technical replicates for each assay condition). Cells were seeded at a density of 25,000 cells / well in 96-well plates (Corning, 356690). Twenty-four hours later, the serum from the cynomolgus macaques was serially diluted at 1:4, 1:8, and 1:16 and then incubated at 37°C with AAV9-CMV-lacZ at 1E+4 vg / cell for 1 h. The neutralized sample was then added to the cells in triplicate and the incubation period was extended to 37°C for 24 h. Absorbance was measured using a β-galactosidase assay kit (Beyotime, RG0036) on a SpectraMax L microplate reader (Molecular Devices). Crab-eating macaques that showed less than 50% inhibition at a 1:4 dilution were selected for in vivo studies.

[0122] rAAV administration

[0123] One-year-old cynomolgus monkeys were anesthetized by intramuscular injection of 1% sodium pentobarbital (80 mg / kg), and 2 mL of rAAV library virus was injected into the medullary cistern using a sterile syringe. The library was injected into four monkeys (two males and two females). Three weeks later, the cynomolgus monkeys were anesthetized by intraperitoneal injection of sodium pentobarbital for tissue collection.

[0124] Organization Collection

[0125] Perfusion of the heart is performed using normal saline. The pleural cavity is incised along the manubrium of the sternum, and the pericardium is opened to expose the heart. The left ventricle is incised at an angle of 30° to 45° with the apex of the heart. The needle is secured, and the blood is rapidly flushed with normal saline. The infusion is complete when the fluid from the right atrial appendage becomes colorless and clear.

[0126] The monkey's skull was opened using a motorized craniotomy saw, the brain was removed and dissected in saline solution. Tissues were collected separately from the parietal, frontal, insula, occipital, temporal, hippocampal, striatum, pons, medulla oblongata, midbrain, cerebellum, thalamus, hypothalamus, corpus callosum, pituitary gland, optic nerve, and olfactory bulb of each hemisphere. Each tissue sample was cut in half and immediately frozen on dry ice, then stored at -80°C for further evaluation (DNA and RNA extraction).

[0127] RNA extraction

[0128] Cut a portion (approximately 0.2g) of each frozen tissue into pieces and homogenize it in Trizol using a BeadBug homogenizer (Homogenizers, Benchmark Scientific, D1032-15, D1032-30, D1033-28). Follow the provided manual. RNA extraction was performed using the RNA Mini Kit (Invitrogen, 12183018A). The purified RNA was dissolved in RNase-free water and stored at -80°C.

[0129] Construction of cDNA libraries

[0130] In a 20 μL reverse transcription reaction containing 1 μg RNA, 10 mM dNTP, 1 μL RNase inhibitor, 0.5 μL Oligo DT, 0.5 μL random primers, 1 μL M-MLV, and 4 μL buffer, the resulting RNA products from each brain region were individually reverse transcribed using M-MLV reverse transcriptase (Promega, M170A) to produce the corresponding viral cDNA library.

[0131] Generation of tissue-specific libraries

[0132] Tissue-specific AAV libraries (“tissue libraries”) are generated by PCR amplification of regions containing 21nt insertions for deep sequencing.

[0133] Organizational enrichment score analysis

[0134] Enrichment scores for each variant of the capsid peptide were calculated to evaluate its biodistribution in different tissues. Specifically, each enrichment score was calculated using the following formula:

[0135]

[0136] The “organization library ratio” is defined as the ratio of AAV variant reads in the organization library to the total reads in the organization library; and the “AAV library ratio” is defined as the ratio of AAV variant reads in the AAV library to the total reads in the AAV library.

[0137] Following the first round of screening as described above, 8,162 variant capsid peptides with enrichment scores greater than 100 were selected from the collected tissues. This was achieved by repeating the process described above. Figure 2 Steps 3-5 of the flowchart shown are used to synthesize the corresponding nucleotide sequences of the selected variants to generate a second-round DNA library (R2 library) for further identification of the best tissue-specific variants for each brain region.

[0138] Example 3. Second round of in vivo screening

[0139] As described above, nucleotide sequences of 8,162 capsid variants selected from the first round of library screening were synthesized and used to construct new plasmid libraries (the second round library; the R2 library). The construction of the second round library was the same as that of the first round library as described earlier, and the library was injected into two cynomolgus monkeys (one female and one male).

[0140] Following the same procedure as the first-round library described in Example 2, in vivo screening and sequence analysis were performed using the second-round AAV library. As shown in Table 1, deep sequencing analysis identified the number of inserted sequences detected in each brain region. L represents the left hemisphere; R represents the right hemisphere.

[0141] Table 1. Number of inserted sequences identified in each tissue

[0142]

[0143] Of the variants identified from the second round of library screening, 60 capsid candidates were selected based on the following two criteria: (1) a high enrichment score in at least one brain region (cutoff value ≥1000); and (2) a higher enrichment score compared to the two reference capsids, AAV9 and PHP.eB. These 60 capsid candidates were named ViVec-N001 to ViVec-N060. Some capsid candidates (ViVec-N001, ViVec-N002, ViVec-N003, and ViVec-N004) showed higher enrichment scores in most brain regions (see [link to relevant documentation]). Figure 3Some candidates (including ViVec-N020, ViVec-N021, ViVec-N023, and ViVec-N033) showed higher enrichment scores in multiple disease-related brain regions (e.g., the frontal and temporal lobes for frontotemporal dementia or the hippocampus for Alzheimer's disease). Some candidates (like ViVec-N005 and ViVec-N006) were particularly enriched in one brain region. The inserted 21 nucleotide and 7 amino acid sequences of these 60 selected capsid variant candidates are shown in Tables 2 and 3, respectively. The complete sequences of the Cap gene and protein can be determined accordingly by inserting individual sequences between amino acid positions 588 and 589 of the wild-type AAV9 sequence as shown in SEQ ID NO:121 (amino acid sequence) and SEQ ID NO:122 (nucleotide sequence).

[0144] Table 2. Insertion 21 nucleotide sequences of 60 selected AAV variants

[0145]

[0146]

[0147] Table 3. Inserted 7-amino acid sequences of 60 selected AAV variants

[0148]

[0149]

[0150] Example 4. In vivo characterization of the selected AAV capsid candidate

[0151] Sixty capsid candidates from the second round of library screening were constructed into a dual plasmid system for AAV packaging, the dual plasmid system comprising a primary plasmid and an auxiliary RC plasmid.

[0152] Preparation of AAV GFP barcode library

[0153] The 60 capsid variants selected from the second round of screening were used to construct the corresponding helper plasmids. AAV was packaged using a two-plasmid system consisting of the main plasmid pAAV-CMV-GFP-lib and the helper plasmids.

[0154] like Figure 4 As shown, a master plasmid was constructed to contain a packaged genome and two ITR elements, each ITR element being side-mounted to a luciferase gene construct operatively linked to the GFP gene via a T2A peptide under the control of a CMV promoter. The construct also includes a barcode downstream of the GFP gene.

[0155] Helper plasmids were constructed to contain the ampicillin resistance gene, AAV2-REP78, Ad5ψ, and other elements involved in AAV capsid translation and assembly, as well as the Cap sequence encoding the AAV capsid protein.

[0156] To construct helper plasmids for different AAV variants, the Cap gene was cut using SwaI and PacI (NEB) in the reaction mixture shown below:

[0157] reagents quantity helper plasmids 12ug SwaI 10uL PacI 10uL Buffer 3.1 10uL <![CDATA[ddH2O]]> Up to 100uL

[0158] The reaction was carried out overnight at 37°C, and the plasmid backbone was recovered after gel electrophoresis. Each candidate capsid coding sequence was ligated into the backbone vector via infusion. After plasmid extraction, the AAV transgenic major plasmid pAAV-CMV-GFP-lib (10 ng) and pUC19 (8 μg) were co-transfected with the AAV helper plasmid (30 μg) into HEK293T cells / 15 cm culture dishes using lipofectamine LTX and plus reagent (Invitrogen, 94756).

[0159] The same AAV packaging process used to generate R1 and R2 AAV libraries was performed to produce AAV variant libraries containing 60 selected capsid variants. After purification and concentration, most of the 60 selected AAV variants were mixed equally, except for a few low-yield AAV variants, to construct the AAV GFP-barcode library.

[0160] Internal research on cynomolgus monkeys

[0161] The AAV library was delivered via intracerebellar medullary injection to cynomolgus monkeys that met our AAV neutralizing antibody (NAb) screening criteria as described in Example 2 above.

[0162] Perform the same tissue collection procedure as for R1 and R2 library screening. Extract genomic DNA (gDNA) from the collected tissue. Following the manufacturer's instructions, use one 30 mg sample of frozen tissue for gDNA extraction using the EZNA MicroElute Genomic DNA Kit (Omega, D3096-02). Determine the concentration and quality of the gDNA using a Qubit 4 fluorometer (Invitrogen, Q33226).

[0163] Tissue enrichment scores of multiple AAV variants

[0164] The vector genome copy number for each variant was determined by deep sequencing of genomic samples processed from collected tissues comprising the parietal, frontal, insula, occipital, temporal, hippocampal, striatum, midbrain, cerebellum, thalamus, hypothalamus, pons, medulla oblongata, and spinal cord. Results were obtained in... Figures 5-18 As shown in the image.

[0165] like Figures 5-18 As shown, the enrichment score of each capsid candidate was normalized relative to the enrichment score of AAV9. ViVec-N001, ViVec-N002, ViVec-N003, ViVec-N004, ViVec-N005, ViVec-N007, ViVec-N008, ViVec-N009, ViVec-N010, ViVec-N011, ViVec-N012, ViVec-N015, ViVec-N016, ViVec-N017, ViVec-N020, ViVec-N021, ViVec-N022, ViVec-N023, ViVec-N024, ViVec-N025, ViVec-N026, ViVec-N027, ViVec-N028, ViVec-N ViVec-N029, ViVec-N030, ViVec-N033, ViVec-N035, ViVec-N036, ViVec-N037, ViVec-N038, ViVec-N039, ViVec-N040, ViVec-N041, ViVec-N043, ViVec-N044, ViVec-N045, ViVec-N046, ViVec-N047, ViVec-N049, ViVec-N050, ViVec-N051, ViVec-N053, ViVec-N054, ViVec-N055, and ViVec-N058 outperformed AAV9 (relative enrichment score >1) in at least one brain region.

[0166] like Figure 5Shown, in the parietal lobe, ViVec-N030, ViVec-N005, ViVec-N022, ViVec-N029, ViVec-N002, ViVec-N033, ViVec-N026, ViVec-N04 3. ViVec-N007, ViVec-N024, ViVec-N008, ViVec-N015, ViVec-N038, ViVec-N004, ViVec-N003, ViVec-N023, ViVe c-N009, ViVec-N040, ViVec-N055, ViVec-N027, ViVec-N054, ViVec-N049, ViVec-N010, ViVec-N044, ViVec-N058, ViVec-N001, ViVec-N039, ViVec-N047, ViVec-N021, ViVec-N016, ViVec-N028, ViVec-N020, and ViVec-N045 are listed from highest to lowest.

[0167] like Figure 6 As shown, in the frontal lobe, ViVec-N030, ViVec-N029, ViVec-N005, ViVec-N033, ViVec-N023, ViVec-N004, ViVec-N002, ViVec-N022, ViVec-N007, ViVec-N008, ViVec-N024, ViVec-N026, ViVec-N015, and ViVec-N040... ViVec-N038, ViVec-N010, ViVec-N054, ViVec-N025, ViVec-N047, ViVec-N009, ViVec-N049, ViVec-N055, ViVec-N020, ViVec-N028, ViVec-N027, ViVec-N001, ViVec-N044, and ViVec-N039 are listed from highest to lowest.

[0168] like Figure 7As shown, in the temporal lobe, ViVec-N029, ViVec-N030, ViVec-N005, ViVec-N033, ViVec-N023, ViVec-N004, ViVec-N002, ViVec-N022, ViVec-N040, ViVec-N007, ViVec-N008, ViVec-N024, ViVec-N015, ViVec-N026, ViVec-N051, ViVec-N038, and ViVec-N003... ViVec-N025, ViVec-N035, ViVec-N054, ViVec-N010, ViVec-N047, ViVec-N009, ViVec-N049, ViVec-N028, ViVec-N055, ViVec-N020, ViVec-N027, ViVec-N050, ViVec-N058, ViVec-N001, ViVec-N039, ViVec-N044, and ViVec-N011 are arranged from highest to lowest.

[0169] like Figure 8 As shown, in the occipital lobe, ViVec-N022, ViVec-N002, ViVec-N005, ViVec-N033, ViVec-N029, ViVec-N007, ViVec-N026, ViVec-N030, ViVec-N008, ViVec-N024, ViVec-N004, ViVec-N015, ViVec-N020, ViVec-N040, ViVe c-N009, ViVec-N038, ViVec-N054, ViVec-N023, ViVec-N049, ViVec-N010, ViVec-N055, ViVec-N047, ViVec-N001, ViVec-N027, ViVec-N044, ViVec-N058, ViVec-N025, ViVec-N028 and ViVec-N039 are arranged from highest to lowest.

[0170] like Figure 9As shown, in the island lobes, ViVec-N030, ViVec-N029, ViVec-N040, ViVec-N005, ViVec-N023, ViVec-N033, ViVec-N004, ViVec-N002, ViVec-N021, ViVec-N022, ViVec-N015, ViVec-N007, ViVec-N024, ViVec-N008, ViVec-N026, ViVec-N038, ViVec-N025, and ViVec-N051 ViVec-N020, ViVec-N035, ViVec-N054, ViVec-N010, ViVec-N047, ViVec-N009, ViVec-N028, ViVec-N041, ViVec-N049, ViVec-N043, ViVec-N027, ViVec-N055, ViVec-N001, ViVec-N050, ViVec-N017, ViVec-N039, ViVec-N044, and ViVec-N011 are arranged from highest to lowest.

[0171] like Figure 10 As shown, in the striatum, ViVec-N030, ViVec-N029, ViVec-N005, ViVec-N021, ViVec-N043, ViVec-N033, ViVec-N004, ViVec-N003, ViVec-N023, ViVec-N022, ViVec-N002, ViVec-N007, ViVec-N024, ViVec-N00 8. ViVec-N015, ViVec-N026, ViVec-N040, ViVec-N009, ViVec-N038, ViVec-N047, ViVec-N020, ViVec-N054, ViVec-N010, ViVec-N016, ViVec-N028, ViVec-N049, ViVec-N041 and ViVec-N055 are listed from highest to lowest.

[0172] like Figure 11As shown, in the hippocampus, ViVec-N005, ViVec-N030, ViVec-N023, ViVec-N033, ViVec-N022, ViVec-N002, ViVec-N004, ViVec-N007, ViVec-N008, ViVec-N024, ViVec-N026, ViVec-N029, ViVec-N021, ViVec-N015, ViVec-N038, and ViVec-N010... ViVec-N009, ViVec-N054, ViVec-N044, ViVec-N055, ViVec-N047, ViVec-N049, ViVec-N040, ViVec-N025, ViVec-N028, ViVec-N027, ViVec-N039, ViVec-N001, ViVec-N058, ViVec-N020, ViVec-N003, and ViVec-N036 are arranged from highest to lowest.

[0173] like Figure 12 As shown, in the midbrain, ViVec-N030, ViVec-N005, ViVec-N029, ViVec-N023, ViVec-N004, ViVec-N033, ViVec-N002, ViVec-N022, ViVec-N007, ViVec-N008, ViVec-N024, ViVec-N015, ViVec-N026, ViVec-N037, ViVec-N047, and ViVec-N040 are arranged from highest to lowest.

[0174] like Figure 13 As shown, in the thalamus, ViVec-N030, ViVec-N029, ViVec-N040, ViVec-N005, ViVec-N033, ViVec-N004, ViVec-N022, ViVec-N002, ViVec-N023, ViVec-N007, ViVec-N026, ViVec-N024, ViVec-N015, ViVec-N015... c-N008, ViVec-N003, ViVec-N025, ViVec-N054, ViVec-N047, ViVec-N009, ViVec-N021, ViVec-N038, ViVec-N010, ViVec-N020, ViVec-N028, ViVec-N049, ViVec-N037 and ViVec-N051 are arranged from high to low.

[0175] like Figure 14As shown, in the hypothalamus, ViVec-N030, ViVec-N003, ViVec-N021, ViVec-N029, ViVec-N005, ViVec-N004, ViVec-N023, ViVec-N033, ViVec-N002, ViVec-N022, ViVec-N007, ViVec-N024, ViVec-N040, ViVec-N008, ViVec-N026, ViVec-N038, ViVec-N040... c-N015, ViVec-N010, ViVec-N025, ViVec-N036, ViVec-N047, ViVec-N054, ViVec-N053, ViVec-N020, ViVec-N017, ViVec-N035, ViVec-N006, ViVec-N009, ViVec-N028, ViVec-N051, ViVec-N012, ViVec-N044, and ViVec-N049 are listed from highest to lowest.

[0176] like Figure 15 As shown, in the cerebellum, ViVec-N030, ViVec-N005, ViVec-N004, ViVec-N023, ViVec-N002, ViVec-N033, ViVec-N022, ViVec-N029, ViVec-N007, ViVec-N026, ViVec-N008, ViVec-N015, ViVec-N024, and ViVec-N021... ViVec-N040, ViVec-N003, ViVec-N054, ViVec-N010, ViVec-N038, ViVec-N025, ViVec-N020, ViVec-N047, ViVec-N009, ViVec-N049, ViVec-N028, ViVec-N055, ViVec-N001, and ViVec-N044 are listed from highest to lowest.

[0177] like Figure 16As shown, in the medulla oblongata, ViVec-N029, ViVec-N005, ViVec-N030, ViVec-N023, ViVec-N040, ViVec-N033, ViVec-N022, ViVec-N004, ViVec-N007, ViVec-N006, ViVec-N002, ViVec-N024, ViVec-N008, and ViVec-N008 are listed. c-N053, ViVec-N015, ViVec-N026, ViVec-N003, ViVec-N038, ViVec-N028, ViVec-N021, ViVec-N047, ViVec-N009, ViVec-N010, ViVec-N025, ViVec-N054, ViVec-N049, and ViVec-N037 are listed from highest to lowest.

[0178] like Figure 17 As shown, in the pons, ViVec-N030, ViVec-N005, ViVec-N023, ViVec-N022, ViVec-N033, ViVec-N002, ViVec-N004, ViVec-N029, ViVec-N007, ViVec-N026, ViVec-N003, ViVec-N024, ViVec-N015, and ViVec-N008... ViVec-N040, ViVec-N021, ViVec-N038, ViVec-N054, ViVec-N020, ViVec-N047, ViVec-N010, ViVec-N009, ViVec-N028, ViVec-N025, ViVec-N006, ViVec-N049, ViVec-N041, and ViVec-N037 are listed from highest to lowest.

[0179] like Figure 18As shown, in the spinal cord, ViVec-N029, ViVec-N033, ViVec-N040, ViVec-N005, ViVec-N030, ViVec-N022, ViVec-N023, ViVec-N002, ViVec-N004, ViVec-N007, ViVec-N015, ViVec-N024, ViVec-N008, ViVec-N026, ViVec-N038, and ViVec-N025... ViVec-N021, ViVec-N003, ViVec-N006, ViVec-N010, ViVec-N009, ViVec-N028, ViVec-N049, ViVec-N047, ViVec-N054, ViVec-N051, ViVec-N020, ViVec-N035, ViVec-N043, ViVec-N046, ViVec-N053, and ViVec-N037 are listed from highest to lowest.

[0180] AAV variants ViVec-N002, ViVec-N003, ViVec-N004, ViVec-N005, ViVec-N006, ViVec-N007, ViVec-N008, ViVec-N021, ViVec-N022, ViVec-N023, ViVec-N024, ViVec-N026, ViVec-N029, ViVec-N030, ViVec-N033, ViVec-N040, and ViVec-N043 ranked in the top 10 in multiple brain regions.

[0181] Example 5. In vitro evaluation of human dopaminergic (DA) neurons

[0182] Human dopaminergic neurons (DA neurons, derived from commercial human embryonic stem cells) were maintained in Neurobasal (Gibco, A1371201), which contained 1% Glutmax (Gibco, A1286001), 2% B27 supplement (Gibco, A3353501), 20 ng / mL BDNF (Peprotech, AF-450-02), 20 ng / mL GDNF (Peprotech, AF-450-10), 1 ng / mL TGFβ3 (Peprotech, AF-100-36E), 200 μM ascorbic acid (Sigma, A4403), 500 μM bibutyryl cAMP (Sigma, D0627-1g) and 10 μM DAPT (Tocris, 2634). DA neurons were seeded at a density of 1.5E+6 cells / well in 12-well plates (Corning, 3513) and allowed to grow in the same medium. The next day, DA neurons were transduced with (1) a GFP-barcoded AAV library containing 60 selected candidate AAV variants prepared as described in Example 4, at a MOI of 1E+4 vg / cell or 1E+5 vg / cell, (2) AAV9 as a reference, or (3) AAV-PHP.eB as another reference. Cultures were collected 72 h after transduction, RNA was extracted, and reverse transcribed into cDNA. AAV capsids in the DA neurons were amplified, and enrichment scores for each AAV variant were determined by deep sequencing.

[0183] To identify rAAVs that exhibit stronger transgene expression and thus improved transduction efficiency compared to AAV9, fold change was calculated as the ratio of the enrichment score of rAAV to that of AAV9. Figure 19 The table shows the log2 (fold change) value for each rAAV calculated based on data obtained from repeated experiments. A log2 (fold change) value higher than 0 indicates a higher expression level. Figure 19As shown, 37 variants exhibited stronger transgenic GFP expression compared to wild-type AAV9. The 37 variants, ranked from highest to lowest enrichment score, are ViVec-N005, ViVec-N010, ViVec-N046, ViVec-N002, ViVec-N028, ViVec-N004, ViVec-N022, ViVec-N025, ViVec-N033, ViVec-N049, ViVec-N007, ViVec-N023, ViVec-N024, ViVec-N008, ViVec-N026, ViVec-N015, ViVec-N040, and ViVec-N03. 8. ViVec-N030, ViVec-N029, ViVec-N054, ViVec-N003, ViVec-N020, ViVec-N047, ViVec-N059, ViVec-N043, ViVec-N009, ViVec-N057, ViVec-N053, ViVec-N001, ViVec-N016, ViVec-N058, ViVec-N012, ViVec-N014, ViVec-N035, ViVec-N055, and ViVec-N017. The first 27 variants even outperformed AAV-PHP.eB, showing stronger transgenic GFP expression.

[0184] Example 6. In vitro validation of the selected AAV capsid candidates individually.

[0185] Preparation of transgenic plasmids and helper plasmids

[0186] The master plasmid pAAV-CMV-Luc-T2A-GFP was constructed by inserting the construct CMV-Luc-T2A-GFP, which contains the target gene (GOI, in this case GFP linked via the T2A peptide and controlled by the CMV promoter) into the plasmid px602 (Addgene). Figure 20 As shown, the plasmid from 5' to 3' contains the following elements: 5' ITR, CMV promoter, GOI (a GFP and luciferase coding sequence linked via T2A), and polyA.

[0187] The RC helper plasmid contains AAV2-REP78, Ad5ψ, and other elements that help translate and assemble the AAV capsid, as well as the Cap sequence encoding the AAV capsid protein that inserts a heptamer between amino acid residues 588 and 589.

[0188] Preparation of AAV variants for individual validation

[0189] To evaluate the transduction capacity of rAAV monovariate capsids into human dopaminergic neurons, a monovariate capsid containing rAAV was prepared. HEK293T cells were co-transfected with the major plasmid pAAV-CMV-Luc-T2A-GFP and an RC helper plasmid containing the monovariate capsid sequence using lipofectamine LTX and plus reagent. AAV particles were harvested after 72 h and purified by iodixanol gradient ultracentrifugation as described above. After determining the viral titer, the purified rAAV was stored at -80°C.

[0190] AAV Quality Control

[0191] AAV genomic titers were determined by qPCR. 20 μL of the rAAV vector was purified by treating with DNase I and proteinase K. The mixture was diluted at ratios of 1:10, 1:100, and 1:1,000. All qPCRs were performed using 2×QuantiFast probe PCR premix and primer / probe sets that specifically bind to the sequence fragment of the luciferase gene.

[0192] Through 10-fold serial dilution (five concentrations, ranging from 10): 12 -10 8 A standard curve for PCR quantification was prepared using plasmid pAAV-CMV-Luc-T2A-GFP (copies / mL). The final reagent concentrations in a 20 μL reaction mixture were: 0.4 μM primer targeting GFP, 0.4 μM probe, 1× ROX reference dye, 2 μL template, and 1× premix. PCR was run in a CFX96Touch real-time PCR detection system (Bio-Rad) under the following conditions: 37°C for 2 min, 95°C for 10 min, followed by 45 cycles at 95°C for 10 s and 62°C for 45 s. Raw data were exported and analyzed using BioRad CFX manager software. Viral titers are given in vg / mL.

[0193] Primers for GFP sequence SEQ ID NO GFP-F8 TCCGCCACAACATCGAGGAC 125 GFP-R8 GTAGTGGTTGTCGGGCAGCA 126 GFP-P8 FAM-CAGCGTGCAGCTCGCCGACC-MGB 127

[0194] For purity control, the purified virus (2×10) 10 (1 particle) is mixed with protein-loaded dye and heated at 95°C for 10 min.

[0195] The samples were then separated on a SurePAGE 4%–12% Bis-Tris gel (GenScript, M00653). After electrophoresis, the gels were stained using the Pierce silver staining kit (Thermo Scientific, 24612) according to the manufacturer's instructions. Briefly, the gels were washed twice with distilled water on a shaker for 5 min each time, then fixed in a fixation solution (water:anhydrous ethanol:acetic acid, volume ratio = 6:3:1) for 15 min and then fixed for 2 h. The gels were washed twice with 10% ethanol and water for 5 min each time. After incubation in 2‰ silver staining sensitizer for 1 min, the gels were rapidly washed twice with water for 1 min each time. After soaking the gels in silver staining solution in the dark for 30 min, silver staining developer was added. Once the target bands appeared, 5% acetic acid was added to terminate the reaction, and images were captured using a Bio-Rad gel imaging system.

[0196] Example 7. In vitro primary neuronal cell evaluation of the selected AAV capsid candidate

[0197] The top four capsid candidates (ViVec-N001, ViVec-N002, ViVec-N004, and ViVec-N023) identified in two rounds of in vivo selection were used to construct helper plasmids (RC helper plasmids) for Rep and Cap, yielding RC-001, RC-002, RC-004, and RC-023. Each specific Cap-containing RC helper plasmid was packaged together with the pAAV-CMV-Luc-T2A-GFP plasmid to generate capsid-specific AAVs for further evaluation.

[0198] Human DAD25 and DAD45 cells were maintained in the culture medium described in Example 5 above and seeded in 12-well plates (Corning, 3513) at a density of 1.5E+6 cells / well. The next day, cells were transduced with AAV particles containing ViVec-N001, ViVec-N002, ViVec-N004, or ViVec-N023 at an MOI of 1E+4 vg / cell or 1E+5 vg / cell. AAV9 and PHP.eB were used as controls. Luciferase levels were measured using a Bright-Lite luciferase assay at 72 hours post-infection, and luminescence intensity was analyzed using a microplate analyzer. The transduction efficiency of individual AAV variants was determined by comparing luminescence intensity with wild-type AAV9.

[0199] like Figure 21A and Figure 21BAs shown, compared with wild-type AAV9, variants ViVec-N001, ViVec-N002, ViVec-N004, and ViVec-N023 exhibited significantly increased luciferase gene expression. Furthermore, ViVec-N002 and ViVec-N004 were superior to AAV-PHP.eB.

[0200] Similarly, four AAV variants were tested in iPSC-derived human cortical neurons. Figure 22 ).

[0201] To test in cortical neurons, AAV capsid variants were prepared by co-transfecting the AAV major plasmid pAAV-CMV-Luc-T2A-GFP (8 μg), RC plasmid (30 μg), and AAV helper plasmid (30 μg) into HEK293T cells / 15cm culture dishes using a three-plasmid system with lipofectamine LTX and plus reagent. Transfected cells and culture medium were harvested after 72 h, followed by centrifugation and purification as described above.

[0202] iPSC-derived cortical neural progenitor cells were purchased from Hopstem Biotechnology and prepared according to the product manual. These progenitor cells were cultured in Matrigel-coated plates, differentiated, and matured for 45 days to obtain cortical neurons. After maturation, AAV variants ViVec-N001, ViVec-N002, ViVec-N023, ViVec-N004, or AAV9 were added to fed medium at MOIs of 1E+4 and 1E+5. Five days after AAV transduction, cells were harvested, and the infection efficiency of each variant was determined by a luciferase assay as described above. Results showed that ViVec-N001 and ViVec-N002 were superior to AAV9 in human cortical neurons.

[0203] The transduction efficiency of the AAV variant was evaluated 21 days post-differentiation in immature iPSC-derived human motor neurons (prepared from human motor progenitor cells, HopStem#). Figure 23 As shown, both ViVec-N001 and ViVec-N002 outperform AAV9.

[0204] In summary, ViVec-N002 demonstrated the best transduction efficiency among these neuronal subtypes other than DA neurons, suggesting its potential to efficiently deliver GOI to different regions of the nervous system for the treatment of a variety of neuronal diseases.

[0205] Other AAV variants were also evaluated in human DA neurons, cortical neurons, and motor neurons.

[0206] Example 8. In vivo validation of the selected AAV capsid candidates individually.

[0207] Several capsid candidates were selected to further evaluate their in vivo biodistribution in the central nervous system.

[0208] ViVec-N001 or ViVec-N004 (5E+12 in 2 ml of medium) carrying the GFP reporter gene were delivered to the brains of cynomolgus monkeys via intracerebellomedullary cistern (ICM) injection (two subjects per capsid). AAV9 was used as a reference. Four weeks after injection, tissues from different regions of the nervous system were collected, and the relative levels of GFP expression were determined by immunostaining.

[0209] Compared to AAV9, both ViVec-N001 and ViVec-N004 showed stronger GFP expression in the striatum. Figure 24A In the hippocampus, ViVec-N001 outperforms AAV9. Figure 24B In the medulla, ViVec-N001 and ViVec-N004 showed GFP expression levels comparable to AAV9. Figure 24C Given that the medulla is the region closest to the injection site, the above observations suggest that the two variants, ViVec-N001 and ViVec-N004, especially ViVec-N001, can spread to more distant regions and likely provide a wider distribution in the nervous system.

[0210] Candidates were further evaluated using different administration routes. ViVec-N002, ViVec-N033, and ViVec-N022 (2.5e12 per animal) carrying luciferase and a GFP reporter gene were delivered to the brains of cynomolgus monkeys via intracerebrospinal injection. AAV9 was used as a reference. Four weeks post-injection, tissues from different regions of the nervous system were collected, and the relative levels of luciferase expression were determined by qPCR (at the DNA and mRNA levels) and immunostaining (at the protein level). Results were presented in... Figures 25-27 As shown in the image.

[0211] Overall, ViVec-N002, ViVec-N033, and ViVec-N022 exhibit a wider and stronger biodistribution than AAV9. Figure 25Compared to AAV9, ViVec-N002 demonstrated higher transduction efficiency in the following regions: frontal cortex (lobe), occipital cortex (lobe), parietal cortex (lobe), temporal cortex (lobe), striatum, midbrain, cerebellum, pons, thalamus, hypothalamus, cervical spinal cord, thoracic spinal cord, optic nerve, cervical dorsal root ganglion, and thoracic dorsal root ganglion. Based on earlier data, ViVec-N033 and ViVec-N022 are expected to perform similarly to ViVec-N002.

[0212] like Figure 26 As shown, ViVec-N022 exhibited higher levels of luciferase DNA (shown as relative fold changes relative to AAV9) in the following regions: occipital cortex (lobe), temporal cortex (lobe), medulla, striatum, midbrain, pons, spinothoracic, spinolumbar, olfactory bulb, and optic nerve, while significantly lower levels were observed in peripheral organs and the dorsal root ganglion (DRG). ViVec-N033 showed a somewhat different distribution pattern, with relatively higher levels of luciferase DNA in the following regions: frontal cortex (lobe), occipital cortex (lobe), temporal cortex (lobe), medulla, midbrain, pons, hypothalamus, cervical spinal cord, thoracic spinal cord, olfactory bulb, optic nerve, and salivary glands. Similar to ViVec-N022, ViVec-N033 also showed lower distribution in peripheral organs and the DRG.

[0213] At the luciferase RNA level, ViVec-N022 was superior to AAV9 in multiple cortical regions (including the occipital cortex, parietal cortex, temporal cortex, and insula), while N033 was superior to AAV9 in all five cortical regions. Furthermore, in the hippocampus, both ViVec-N022 and ViVec-N033, especially ViVec-N033, expressed more luciferase RNA. The hippocampus is a brain region that plays a crucial role in memory formation and is often the first brain region damaged in Alzheimer's disease patients. Figure 27 Based on the above data, the ViVec AAV capsid tested in this application should be a more effective vector than wild-type AAV9 for intraventricular gene delivery (especially for delivering one or more target genes into the hippocampus to treat memory-related disorders).

[0214] The candidates were further evaluated via intrastriatal administration. ViVec-N001 and ViVec-N002, carrying the GFP reporter gene, were delivered to the mouse striatum via intrastriatal injection (2e12 vg at each injection site). Figure 28As shown, the striatal region transduced by ViVec-N002 is more extensive than that transduced by AAV9. ViVec-N033 and ViVec-N022 were also tested via intrastriatal administration. 1.2E+10 vg of ViVec-N033 and ViVec-N022 were injected into the striatum of mice via intrastriatal injection. Striatal tissue was collected 21 days post-injection, and luciferase RNA levels were determined using RT-qPCR. We found that both ViVec-N033 and ViVec-N022 delivered more luciferase (GOI) compared to AAV9, similar to ViVec-N002.

[0215] The ability of individual candidates to deliver genes to the central nervous system via intrathecal administration was also evaluated. ViVec-N033 and ViVec-N022, carrying both luciferase and the GFP reporter gene, were injected intrathecally into SD rats (two doses, low: 1E+13 vg / kg and high: 3E+13 vg / kg). AAV9 was used as a reference. Luciferase RNA levels in the spinal cord and cortical tissues were determined by RT-qPCR 21 days post-injection. Figure 30 As shown, both ViVec-N033 and ViVec-N022 have better transfection efficiency than AAV9, and express more luciferase RNA in the thoracic spinal cord, lumbar spinal cord (injection site), and cortex.

[0216] The candidates were further evaluated via intravenous administration to test the ability of these novel capsids to cross the blood-brain barrier. ViVec-N002, ViVec-N033, and ViVec-N022, each carrying both luciferase and the GFP reporter gene, were delivered intravenously to two different strains of mice. Three weeks after injection, tissues from different regions of the nervous system and peripheral organs were collected, and the relative levels of luciferase and GFP expression were determined by qPCR (at the DNA and mRNA levels) and immunostaining (at the protein level).

[0217] Wild-type AAV9 VP1 polypeptide sequence (SEQ ID NO: 121; Q588 and A589 are underlined)

[0218] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSA QA QAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

Claims

1. An engineered adeno-associated virus (AAV) capsid polypeptide, wherein the engineered AAV capsid polypeptide has an insertion of any amino acid sequence as shown in SEQ ID NOs: 1, 2, 4, 22, 23, and 33 between amino acid positions 588 and 589 of the AAV9 capsid polypeptide. The amino acid sequence of the capsid polypeptide of AAV9 is shown in SEQ ID NO:

121. The engineered AAV capsid polypeptide can be assembled into recombinant AAV (rAAV) viral particles, and compared with rAAV viral particles with a capsid of serotype AAV9, the rAAV viral particles have increased tropism for one or more tissues or cells of the central nervous system (CNS) and / or can produce higher levels of transgenic expression in tissues or cells of the central nervous system.

2. A recombinant adeno-associated virus (rAAV) viral particle, said recombinant adeno-associated virus viral particle comprising a capsid assembled from the AAV capsid polypeptide according to claim 1 and a vector genome containing transgenes.

3. The rAAV viral particle according to claim 2, wherein the transgene is a therapeutic gene or a reporter gene.

4. A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide according to claim 1.

5. The nucleic acid molecule according to claim 4, wherein the nucleic acid molecule comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 61, 62, 64, 82, 83 and 93.

6. A recombinant adeno-associated virus (rAAV) helper vector, said recombinant adeno-associated virus helper vector comprising a nucleotide sequence encoding the polypeptide according to claim 1.

7. A pharmaceutical composition comprising rAAV viral particles as described in claim 2 or claim 3 and a pharmaceutically acceptable carrier.

8. Use of the rAAV viral particles according to claim 2 or claim 3, or the pharmaceutical composition according to claim 7, in the manufacture of a medicament for treating diseases of the central nervous system (CNS).

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