Novel AAV capsid for targeting nervous system and uses thereof

Abstract

Provided are novel adeno-associated virus (AAV) capsids, as well as recombinant AAV (rAAV) virions comprising the capsids and a vector genome comprising a transgene. The rAAV virions comprising the novel capsid exhibit improved CNS-targeted infection efficiency and / or increased expression levels of the transgene. Also provided are methods for treating a disease comprising administering to a subject in need thereof an rAAV virion comprising the AAV capsid.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefits of PCT application number PCT / CN2022 / 107543, filed on July 22, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present 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 the capsids. rAAV particles comprising novel capsids show improved central nervous system (CNS) targeted transduction efficiency and / or increased expression levels of transgenes.

[0004] Sequence Listing

[0005] This disclosure includes a Sequence Listing as a part of this disclosure. Background Art

[0006] The statements in this section merely provide background information related to the present disclosure and may not 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 show 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 gene therapy success--a personal perspective. Hum Gene Ther, 2015. 26 (5): p. 257-65).

[0008] The AAV genome is a single-stranded DNA (ssDNA) of about 4.7 kb, which has an inverted terminal repeat (ITR) sequence of about 145 bases at both ends. The ITR sequence forms a Watson-Crick base pair to form a T-shaped hairpin structure, which contains cis elements necessary for replication and packaging of the AAV genome. The AAV genome contains two open reading frames (ORFs) in the region flanked by ITR sequences. One ORF (also referred to as a "rep gene") encodes four Rep proteins (Rep78, Rep68, Rep52, and Rep40). Another ORF (also referred to as a "cap gene") encodes three capsid proteins (VP1, VP2, and VP3) and an assembly activation protein (AAP). The Rep protein has helicase activity and is not only required for inducing capsid formation, but also required for integrating the AAV genome into the host cell chromosome. The AAV capsid is formed by 60 molecules of VP1, VP2, and VP3 assembled to form an icosahedral AAV shell at a ratio of 1:1:10. VP1, VP2 and VP3 are mainly different in the N-terminal region, wherein the N-terminal of VP1 contains a unique phospholipase A2 (PLA2) domain. Since the PLA2 domain is only present in VP1, the N-terminal region of VP1 is also referred to as the VP1 unique region (VP1u). It is known that the PLA2 domain is exposed to the external environment of the AAV particles under acidic conditions, but it is usually present inside the AAV particles. Therefore, it is believed that after AAV enters the cell, the PLA2 domain is necessary for AAV to escape from the endosome and translocate into the nucleus. Assembly activation protein (AAP) is a protein necessary for the formation of AAV capsid. The replication of AAV in nature depends on the presence of helper viruses (such as adenovirus and herpes virus). In the presence of helper viruses, the AAV genome is replicated in the host cell to form complete AAV particles containing the AAV genome. Then, the AAV particles are released from the host cell. In the absence of a helper virus, the AAV genome can be maintained in an episomal manner, or less frequently, integrate 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): p. 255-272.).

[0009] AAV can infect a variety of cells, dividing cells and non-dividing cells after mitosis, such as blood cells, muscle cells and nerve cells. Since AAV is not pathogenic to humans, it has a low risk of adverse effects. In addition, AAV virus particles are physicochemically stable. Therefore, AAV has recently attracted 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): Page 767.). For example, AAV9 carrying a functional SMN gene has been developed to treat spinal muscular atrophy (Novartis, Zolgensma). Based on the contained capsid, AAV serotypes are different in their tropism, and therefore can be used to target different tissues and cell types for gene delivery. However, gene delivery by naturally occurring AAV serotypes is limited by dose-limiting safety constraints and their broad tissue tropism, restricting the development of effective AAV-based gene therapies, especially for neurological disorders.

[0010] To expand the AAV toolbox for nervous system delivery, AAV capsid engineering has been performed by rational design (Lee, EJ, CM 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 neural tropism, a peptide insertion strategy was applied to identify AAV-PHP.B and AAV-PHP.eb derived from AAV9, a natural serotype that can cross the blood-brain barrier and infect neural cells (Ravindra Kumar, S. et al., Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. Nat Methods, 2020. 17(5): pp. 541-550).

[0011] In addition, for the treatment of different neurological disorders, AAV serotypes with disease-related neuron-specific tissue tropism will be more desirable for safety and efficacy considerations. For example, AAV with frontal and temporal cortex tropism will be the preferred delivery tool for the treatment of frontotemporal dementia. Other potential strategies may involve the use of AAVs targeting the dopaminergic system in the striatum and midbrain to treat Parkinson's disease, the use of AAVs targeting motor neurons to treat spinal muscular atrophy (SMA) or amyotrophic lateral sclerosis (ALS), and the use of 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 the present invention designed and generated 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 stretch of 7 amino acids (7-mer) at a site between amino acid position 588 and amino acid position 589 of the AAV9 capsid polypeptide (see Figure 1 ).

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

[0015] (1) Ability to assemble into AAV particles,

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

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

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

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

[0020] In a fourth aspect, the present application relates to a recombinant vector comprising the nucleic acid of the third aspect.

[0021] In a fifth aspect, the present application relates to a host cell comprising 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) the AAV capsid and vector genome of the first aspect; or the rAAV particle of the second aspect, and (2) a pharmaceutically acceptable carrier.

[0023] In a seventh aspect, the present invention relates to the use of the AAV capsid polypeptide, the nucleic acid, the rAAV particle or the pharmaceutical composition in the manufacture of a medicament for delivering a transgene to a subject. In a more specific embodiment, the drug 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 a therapeutically effective amount of the rAAV particle of the second aspect or the pharmaceutical composition of the sixth aspect to a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0027] Figure 2 A scheme 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 capsid, PHP-eB capsid and 10 engineered capsids of the present application (candidates ViVec-N001, ViVec-N002, ViVec-N003, ViVec-N004, ViVec-N005, ViVec-N006, ViVec-N020, ViVec-N021, ViVec-N023 and ViVec-N033) in different brain regions are shown. The first column indicates the sequence of the inserted amino acids.

[0029] Figure 4 The structure of the construct pAAV-CMV-GFP-lib to be included in the master plasmid for preparation of the rAAV GFP-barcode library is shown.

[0030] Figure 5-Figure 18 Relative enrichment scores of engineered capsid polypeptides in different CNS tissues are shown.

[0031] Fig.19 Shown are the expression levels of the GFP transgene delivered by rAAV of each engineered capsid polypeptide.

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

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

[0034] Fig. 22 Shown are the results of in vitro validation of four AAV capsid candidates in cultured human cortical neurons (45 days after differentiation) at MOI = 1E+4 or 1E+5.

[0035] Fig.23 Shown are the results of in vitro validation of four AAV capsid candidates in cultured human motor neurons (21 days after differentiation) at MOI = 1E+4 or 1E+5.

[0036] Figure 24A-Figure 24C The 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) from monkeys treated with AAV capsid candidates is shown.

[0037] Fig.25 Biodistribution of ViVec-N002 in the brain and spinal cord of cynomolgus monkeys 21 days after intracerebroventricular (icv) injection is shown. Relative DNA levels of luciferase in each region are shown as fold change (normalized to AAV9-treated group).

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

[0039] Fig. 27 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 to the AAV9-treated group), N=2.

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

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

[0042] Fig.30 Shown are relative RNA levels of luciferase (normalized to the AAV9-treated group) in rat spinal cord and cortex (N=8, 4 males and 4 females) 21 days after intrathecal injection of ViVec-N022, ViVec-N033, and AAV9. DETAILED DESCRIPTION

[0043] definition

[0044] "AAV" refers to adeno-associated virus. "rAAV" refers to recombinant adeno-associated virus. In the context of this application, unless otherwise specified, "rAAV vector", "rAAV virion" and "rAAV viral particle" have the same meaning and refer to a viral particle containing a vector genome encapsulated in a rAAV capsid. The vector genome of rAAV lacks the rep and cap genes in the wild-type AAV genome, so rAAV is a replication-defective entity.

[0045] The "capsid" is formed by assembling three isoforms of "capsid protein" and "capsid polypeptide", which are VP1, VP2 and VP3. "Capsid protein" or "capsid polypeptide" generally does not indicate the type of isoform, and is understood to include all three isoforms.

[0046] The term "engineered capsid polypeptide" refers to an artificially modified capsid polypeptide that contains at least one amino acid difference compared to the wild-type capsid polypeptide of AAV. In a specific embodiment, the engineered capsid polypeptide of the present application has a 7-mer insertion at a position corresponding to the position 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 the present application shows higher tissue tropism in one or more CNS tissues compared to the wild-type AAV9 capsid polypeptide. The numbering and insertion position of the amino acids are based on the VP1 of the wild-type AAV9, because VP1 is the longest isoform of the three capsid proteins. Both VP2 and VP3 are N-terminal truncated forms of VP1, while still containing a region corresponding to the insertion position between Q588 and A589 of the AAV9 VP1 protein. It should be understood that in the case 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 numbering of the insertion positions (between Q451 and A452 in VP2 and between Q386 and A387 in VP3) should be adjusted accordingly.

[0047] The term "gene" refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) involved in encoding an RNA transcript and its corresponding nucleotide sequence. As used herein, the phrase "gene of interest" or its abbreviation "GOI" refers to a gene to be delivered by rAAV. In some embodiments, the GOI comprises only the coding sequence of a polypeptide. In other embodiments, in addition to the coding sequence, the GOI also comprises a non-coding region, a regulatory region. In some embodiments, the GOI does not encode a polypeptide.

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

[0049] The terms "peptide," "polypeptide," or "protein," used interchangeably herein, 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, "variants" refer to polypeptides that differ from wild-type polypeptides by one or more amino acid variations, but are related to wild-type polypeptides in structure, activity and / or function. 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 viral packaging system that uses co-transfection of two plasmids, one plasmid encoding the transgene and the other plasmid encoding the Rep gene, the Cap gene, and the Ad helper genes.

[0053] The term "subject" as used herein 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 a monkey). The term "subject" may also encompass cells, tissues, and progeny of biological entities obtained by in vivo or in vitro culture.

[0054] The phrase "effective amount" or "therapeutically effective amount" refers to an amount of a composition (e.g., a composition comprising rAAV virions) sufficient to result in a desired activity when delivered to a subject in need thereof. The desired activity can encompass delaying the manifestation of a disorder, preventing or delaying the progression of a disorder, or alleviating at least one symptom of a disorder.

[0055] Methods for engineering capsid polypeptides

[0056] The method for preparing the engineered capsid polypeptide of the present application is Figure 2 Briefly presented in . In short, it is carried out by constructing a DNA library containing polynucleotide sequences encoding individual variant capsid polypeptides, each variant capsid polypeptide having a unique 7-mer insertion at a desired position of the AAV9 capsid polypeptide. The types of 7-mer insertions included in the library should theoretically cover all possible amino acid combinations, which number is approximately 20^7. In addition, a plasmid library can be established based on the DNA library of capsid coding sequences and used to express variant capsid polypeptides in host cells to form a library of AAV virus particles. The resulting AAV virus particle library can be delivered to an animal (e.g., a monkey) to be screened for tissue tropism.

[0057] "DNA library" refers to a library of variant polynucleotide sequences encoding variant capsid polypeptides. In particular, the variant polynucleotide sequence comprises a wild-type AAV9 capsid polypeptide sequence with seven NNK codon insertions corresponding to 7-mer amino acid insertions. The NNK codon can encode one of 20 amino acids at the insertion site. This method is called "site saturation mutagenesis (SSM)", which is a powerful tool in protein engineering. The "NNK saturation mutagenesis strategy" is one of the most popular SSM strategies using NNK degenerate codons. The NNK codon can encode one of 20 amino acids, and the chance of encoding a stop codon is small.

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

[0059] "AAV library" or "AAV virion library" refers to a collection of AAV virions formed by capsids expressed by the plasmid library of the present application in a host cell line. For example, the host cell line can 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 a test animal injected with the AAV library. In particular, reverse transcription is performed to generate total cDNA from RNA extracted from the tissue, and viral DNA is amplified from the total DNA to generate a tissue-specific AAV library.

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

[0062] As used herein, "CNS tropism" includes tropism of rAAV for one or more of 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 polypeptides and polynucleotides encoding the same

[0064] In one aspect, the present application provides a series of variant AAV capsid polypeptides. Compared with rAAV particles formed by the capsid of wild-type AAV9, the rAAV particles formed by the variant AAV capsid polypeptides of the present application show increased tropism for one or more tissues or cells of the central nervous system.

[0065] The variant AAV capsid polypeptide of the present application with improved CNS tissue tropism compared to wild-type AAV9 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. In some embodiments, the variant AAV capsid polypeptide of the present application 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 selected from the amino acid sequences shown in SEQ ID NO: 1-60.

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

[0067] AAV library

[0068] In another aspect, the present application provides an AAV library, comprising a plurality of variant AAV capsid polypeptides, 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 one of 20 amino acids. The AAV library has at least 1×10 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 Types of variant capsid polypeptides.

[0069] In another aspect, the present application provides a DNA or plasmid library comprising a plurality of nucleotide sequences encoding variant AAV capsid polypeptides, 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. The DNA or plasmid library can express at least 1×10 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 In a specific embodiment, the nucleotide sequences in the DNA or plasmid library are generated by the NNK saturation mutagenesis strategy by inserting 7 NNK codons between the codons of Q588 and A589 in the wild-type AAV9 VP1 capsid protein coding sequence.

[0070] DNA libraries, plasmid libraries, and AAV libraries as described above can be used to screen for new variant AAV capsid polypeptides or coding sequences having desired properties (such as expression efficiency, tissue tropism, etc.).

[0071] rAAV virus particles

[0072] On the one hand, the present application provides rAAV virus particles comprising a capsid and a vector genome, wherein the capsid comprises an engineered capsid polypeptide of the present application. The rAAV virus particles show tropism in one or more CNS tissues. More preferably, the rAAV virus particles show higher tropism and / or higher transduction efficiency in one or more CNS tissues compared to rAAV virus particles with wild-type AAV9 capsids.

[0073] For example, in one or more CNS tissues, rAAV virions comprising the engineered capsid polypeptides of the present application show at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or 1000% higher transduction efficiency than rAAV virions having rAAV virions comprising wild-type AAV9 capsids. For example, the transduction efficiency of rAAV in the target tissue can be measured and calculated based on the expression level of the 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 the present application shows tissue tropism in one or more brain regions or CNS tissues, and the brain regions or CNS tissues are selected from the parietal lobe, frontal lobe, temporal lobe, occipital lobe, 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 with wild-type capsid of AAV9, rAAV with variant AAV capsid of the present application can show improved tropism in one or more of the brain regions or CNS tissues. rAAV with variant AAV capsid of the present application can lead to the desired distribution and / or expression of GOI in one or more brain regions or CNS tissues, and the brain regions or CNS tissues are selected from the parietal lobe, frontal lobe, temporal lobe, occipital lobe, insula, striatum, hippocampus, midbrain, thalamus, hypothalamus, cerebellum, medulla oblongata, pons, spinal cord, optic nerve, cervical dorsal root ganglion and thoracic dorsal root ganglion. Preferably, the rAAV having the variant AAV capsid of the present application can result in increased expression of the GOI in one or more of the brain regions or CNS tissues compared to the rAAV having the wild-type capsid of AAV9.

[0075] In a specific embodiment, the rAAV virus particle of the present application comprises 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 NOs: 1-60 into a 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 NOs: 1, 2, 4, 22 and 33. In a more preferred embodiment, the 7-amino acid insertion is selected from any one of SEQ ID NOs: 2, 22 and 33. In the context of the present application, rAAV having a capsid comprising a 7-amino acid insertion of SEQ ID NOs: 1-60 is designated as ViVec-N001 to ViVec-N060, respectively.

[0076] In one embodiment, the present application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the parietal lobe. Preferably, the rAAV exhibiting tropism for the parietal lobe comprises a variant AAV capsid polypeptide comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: 129, 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. In particular, the rAAV shows improved tropism to the parietal lobe compared to the rAAV with the wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for the rAAV comprising a variant AAV capsid polypeptide and showing tropism to the parietal lobe, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The present application also relates to the use of the rAAV as described in this paragraph for delivering GOI to the parietal lobe.

[0077] In one embodiment, the present 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 comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: 3 NO:1, SEQ ID NO:44 and SEQ ID NO:39. In particular, the rAAV shows improved tropism for the frontal lobe compared to the rAAV with the wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for the rAAV comprising a variant AAV capsid polypeptide and showing tropism for the frontal lobe, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The present application also relates to the use of the rAAV as described in this paragraph for delivering GOI to the frontal lobe.

[0078] In one embodiment, the present application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting a tropism for the temporal lobe. Preferably, the rAAV exhibiting tropism for the temporal lobe comprises a variant AAV capsid polypeptide comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: 5 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. In particular, the rAAV shows improved tropism for the temporal lobe compared to the rAAV with the wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for the rAAV comprising a variant AAV capsid polypeptide and showing tropism for the temporal lobe, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The present application also relates to the use of the rAAV as described in this paragraph for delivering GOI to the temporal lobe.

[0079] In one embodiment, the present application provides a 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 comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: NO:58, SEQ ID NO:25, SEQ ID NO:28 and SEQ ID NO:39. In particular, the rAAV shows improved tropism for the occipital lobe compared to the rAAV with the wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for the rAAV comprising a variant AAV capsid polypeptide and showing tropism for the occipital lobe, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The present application also relates to the use of the rAAV as described in this paragraph for delivering GOI to the occipital lobe.

[0080] In one embodiment, the present application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the insula. Preferably, the rAAV exhibiting tropism for the insula comprises a variant AAV capsid polypeptide comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: 39, SEQ ID NO: 40 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. In particular, the rAAV exhibits improved tropism for the insula compared to the rAAV with the wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for the rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the insula, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The present application also relates to the use of rAAV as described in this paragraph for delivering GOI to the insula.

[0081] In one embodiment, the present application provides rAAV comprising a variant AAV capsid polypeptide and showing tropism for the striatum. Preferably, the rAAV exhibiting tropism for the striatum comprises a variant AAV capsid polypeptide comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: 39, SEQ ID NO: 49, SEQ ID NO: 50 NO:49, SEQ ID NO:41, SEQ ID NO:55 and SEQ ID NO:1. In particular, the rAAV midbrain shows improved tropism for the striatum compared to the rAAV with the wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for the rAAV comprising a variant AAV capsid polypeptide and showing tropism for the striatum, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The application also relates to the use of the rAAV as described in this paragraph for delivering GOI to the striatum.

[0082] In one embodiment, the present application provides rAAV comprising a variant AAV capsid polypeptide and showing tropism for the hippocampus. Preferably, the rAAV exhibiting tropism for the hippocampus comprises a variant AAV capsid polypeptide comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: 3 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. In particular, rAAV shows improved tropism for hippocampus compared to rAAV with wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for rAAV comprising variant AAV capsid polypeptide and showing tropism for hippocampus, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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, SEQID NO:24 and SEQ ID NO:1. The present application also relates to the use of rAAV as described in this paragraph for delivering GOI to hippocampus.

[0083] In one embodiment, the present application provides a rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the midbrain. Preferably, the rAAV exhibiting tropism for the midbrain comprises a variant AAV capsid polypeptide comprising 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 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, the rAAV exhibits improved tropism for the midbrain compared to the rAAV having the wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for rAAV comprising a variant AAV capsid polypeptide and showing tropism for the midbrain, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The present application also relates to the use of the rAAV as described in this paragraph for delivering GOI to the midbrain.

[0084] In one embodiment, the present application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the thalamus. Preferably, the rAAV exhibiting tropism for the thalamus comprises a variant AAV capsid polypeptide comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: 5 NO:37 and SEQID NO:51. In particular, the rAAV shows improved tropism for the thalamus compared to the rAAV with the wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for the rAAV comprising a variant AAV capsid polypeptide and showing tropism for the thalamus, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The present application also relates to the use of the rAAV as described in this paragraph for delivering GOI to the thalamus.

[0085] In one embodiment, the present application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for the hypothalamus. Preferably, the rAAV exhibiting tropism for the hypothalamus comprises a variant AAV capsid polypeptide comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: 28, SEQ ID NO: 39, SEQ ID NO: 40 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. In particular, rAAV shows improved tropism for the hypothalamus compared to rAAV with wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for rAAV comprising a variant AAV capsid polypeptide and showing tropism for the hypothalamus, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The present application also relates to the use of rAAV as described in this paragraph for delivering GOI to the hypothalamus.

[0086] In one embodiment, the present application provides rAAV comprising a variant AAV capsid polypeptide and showing tropism for the cerebellum. Preferably, the rAAV exhibiting tropism for the cerebellum comprises a variant AAV capsid polypeptide comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: 39, SEQ ID NO: 40 NO:55, SEQ ID NO:1 and SEQ ID NO:44. In particular, the rAAV shows improved tropism for the cerebellum compared to the rAAV with the wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for the rAAV comprising a variant AAV capsid polypeptide and showing tropism for the cerebellum, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The present application also relates to the use of the rAAV as described in this paragraph for delivering GOI to the cerebellum.

[0087] In one embodiment, the present application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting a tropism for the medulla. Preferably, the rAAV exhibiting tropism for the medulla comprises a variant AAV capsid polypeptide comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: 26 NO:49, SEQ ID NO:37 and SEQ ID NO:1. In particular, the rAAV shows improved tropism for the medulla compared to the rAAV with the wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for the rAAV comprising a variant AAV capsid polypeptide and showing tropism for the medulla, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The present application also relates to the use of the rAAV as described in this paragraph for delivering GOI to the medulla.

[0088] In one embodiment, the present 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 comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: 7 NO:49, SEQ ID NO:41 and SEQ ID NO:37. In particular, the rAAV shows improved tropism for the pons compared to the rAAV with the wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for the rAAV comprising a variant AAV capsid polypeptide and showing tropism for the pons, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The present application also relates to the use of the rAAV as described in this paragraph for delivering GOI to the pons.

[0089] In one embodiment, the present application provides rAAV comprising 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 comprising a 7 amino acid insertion between amino acid position Q588 and amino acid position A589 of the wild-type AAV9 VP1 capsid protein as set forth in SEQ ID NO: 121 as set forth 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: 55 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. In particular, rAAV shows improved tropism for the spinal cord compared to rAAV with wild-type capsid of AAV9 or AAV-PHP.eB. More preferably, for rAAV comprising a variant AAV capsid polypeptide and showing tropism for the spinal cord, the variant AAV capsid polypeptide comprises a 7 amino acid insertion as shown in any one 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. The present application also relates to the use of rAAV as described in this paragraph for delivering GOI to the spinal cord. The spinal cord can be a cervical spinal cord and / or a thoracic spinal cord.

[0090] In one embodiment, the present application provides rAAV comprising a variant AAV capsid polypeptide and exhibiting tropism for dopaminergic (DA) neurons. Preferably, the rAAV that exhibits tropism for DA neurons comprises a variant AAV capsid polypeptide comprising a 7 amino acid insertion as set forth in any one of 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, SEQ ID NO:2 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 the rAAV having the variant AAV capsid polypeptide shows improved tropism for DA neurons compared to the rAAV having the wild-type capsid of 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 the rAAV having the variant AAV capsid polypeptide shows improved tropism for DA neurons compared to the rAAV having the wild-type capsid of AAV-PHP.eB. The present application also relates to the use of the rAAV as described in this paragraph for delivering GOI to DA neurons.

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

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

[0093] use

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

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

[0096] The rAAV of the present application can be administered via different routes. For example, the rAAV of the present application can be administered systemically or locally to a subject. For example, the route of administration includes, but is not limited to, intravenous route, intramuscular route, intracisternal injection, intrathecal injection, intravitreal injection, intraparenchymal injection, or intraventricular injection.

[0097] Example

[0098] Example 1. Preparation of AAV capsid library

[0099] By using the NNK saturation mutagenesis strategy adapted from the prior art (Ravindra Kumar, S. et al., supra), a mutagenized AAV library containing a mutant Cap gene (VP1 gene, the coding sequence of the wild-type AAV9 capsid protein VP1 is shown in SEQ ID NO: 122) was generated for the first round of screening (Round 1 library, or "R1 library"). The mutant capsid coding sequence contained in the Round 1 library has a random 21-base stretch consisting of 7-NNK codons, thereby encoding a mutant capsid protein having a heptamer (7 amino acids or 7-mers), which is inserted 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 one of A, C, G and T, and K can be G or T. Therefore, the entire library can cover all possible 7 amino acid combinations (see Figure 1 ). The construction of the first round of libraries is described in detail below. A brief description of the workflow is in Figure 2 Shown in.

[0100] Synthesis of random fragments

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

[0102] Digestion of the skeleton

[0103] The vector (rAAV-Cap-in-cis) was linearized using KpnI and AgeI (NEB), and the region encoding the amino acids from position 546 (inclusive) to position 599 (inclusive) of the AAV9 VP1 protein was removed. The enzyme digestion mixture is shown below.

[0104] Reagents quantity Carrier 12ug KqI 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 by Zymoclean Gel DNA Recovery Kit (ZYMORESEARCH, D4008).

[0106] connect

[0107] At 50°C, random library fragments were HiFi DNA Assembly Master Mix (NEB, E2621) was ligated into the linearized backbone for 1 h to generate the R1 library plasmid (rAAV-CapLib-in-cis).

[0108] Plasmid transformation

[0109] After ligation, the mixture was diluted 4 times, and 10uL of the diluted ligation mixture was added to 100uL of TransStbl3 chemical competent cells (TransGen Biotech, CD521-02), and incubated on ice for 30min, then heated at 42°C for 30s, and placed on ice for 3min. Afterwards, 1mL of SOC medium was added to the cells and incubated at 37°C for 2h in a shaker. The cells were then transferred to 20mL of LB medium containing 50ug / mL ampicillin and incubated at 37°C for 4h, and then transferred to 300mL of LB medium containing 100ug / mL ampicillin for continued growth for 12h, and then the plasmid was extracted.

[0110] Plasmid extraction

[0111] According to the manufacturer's instructions, the plasmid for rAAV production was extracted using the Purelink hipure filter maxi kit (Thermo, K210018). In short, 300mL overnight culture was centrifuged at 8,000rpm to collect bacteria, and resuspended with 15mL R3, followed by the addition of 15mL L7 to lyse for 5min. Then 15mL N3 was added for 10min and centrifuged at 12,000rpm, and the supernatant was then poured into a column treated with EQ1. Then 40mL W8 was added to wash the column twice, followed by the addition of 15mL E4 to elute the plasmid DNA into a new centrifuge tube. 10.5mL isopropanol was added to the tube, and centrifuged at 9,000rpm for 1h at 4°C to collect plasmid DNA. The supernatant was removed, and the precipitated plasmid DNA was transferred to a 1.5mL centrifuge tube, resuspended, and washed twice with 70% ethanol. The tubes were then placed at 65°C to allow the ethanol to evaporate, and 200 uL of sterile water was added to dissolve the plasmid DNA.

[0112] Production and purification of rAAV

[0113] HEK293T cells were cultured in Dulbecco's modified Eagle's 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 production, rAAV-CapLib-in-cisDNA library was co-transfected with AAV helper plasmid into HEK293T cells in 15 cm culture dishes using lipofectamine LTX and plus reagent (Invitrogen, 94756). After incubation for 6-8 h, the culture medium was replaced with DMEM (5% FBS, 1% PS). The first collection was performed by collecting rAAV from the supernatant 48 h after transfection. The cells were harvested for the second time with the culture medium 72 h after transfection.

[0115] For iodixanol purification, HEK293T cells from eight 15cm plates were dissolved 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 15% phase with a final concentration of 1M. In order to more easily distinguish the interface in the gradient, 2 μL phenol red was added to 25% and 60% iodixanol solution of 1mL respectively. 15mL processed cell lysates, 9mL 15%, 6mL 25%, 5mL 40% and 5mL 60% iodixanol solution were added to 39mL Quick-Seal tubes (Beckman, 344326) from top to bottom. After centrifugation at 60,000 rpm in a 70Ti rotor for 1.5 h at 18°C, rAAV was collected from the 40% phase and washed with 0.001% Pluronic TM F-68 nonionic surfactant (Gibco, 24040032) in PBS was ultrafiltered and concentrated using an Amicon Ultra-4 centrifugal filter (Millipore, UFC810096). The purified rAAV was stored at -80°C as the R1 rAAV library.

[0116] R1 library characterization

[0117] Both the R1 DNA library rAAV-CapLib-in-cis and the final R1 rAAV library were deeply sequenced. In the R1 rAAV library prepared from the R1 DNA library, 376,441 types of unique insert sequences were confirmed.

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

[0119] This example 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] Screening of AAV-naïve monkeys by in vitro neutralizing antibody assay

[0121] AAV neutralizing antibodies (NAbs) in experimental animals were determined via in vitro neutralizing antibody assays to identify and select monkeys that were not treated with AAV for screening rAAV libraries. Serum from 1-year-old cynomolgus macaques was collected 1 month before injection (n=3 technical replicates for each assay condition). Cells were seeded in 96-well plates (Corning, 356690) at a density of 25,000 cells / well. Twenty-four hours later, serum from cynomolgus macaques was serially diluted at 1:4, 1:8, and 1:16, and then incubated with AAV9-CMV-lacZ at 1E+4vg / cell for 1h at 37°C. The neutralized samples were then added to the cells in triplicate and returned to 37°C for 24h. The absorbance was measured on a SpectraMax L microplate reader (Molecular Devices) using a β-galactosidase assay kit (Beyotime, RG0036). Cynomolgus monkeys showing less than 50% inhibition at a dilution of 1:4 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 cisterna magna of the medulla oblongata with a sterile syringe. The library was injected into four monkeys (two males and two females). Three weeks later, cynomolgus monkeys were anesthetized by intraperitoneal injection with sodium pentobarbital for tissue collection.

[0124] Tissue collection

[0125] The heart is perfused with normal saline. The thorax is incised along the manubrium of the sternum, and the pericardium is incised to expose the heart. The left ventricle is incised at an angle of 30° to 45° to the apex of the heart. The needle is fixed, and the blood is then quickly flushed with normal saline, and the infusion is complete when the fluid from the right atrial appendage becomes colorless and transparent.

[0126] The skull of monkey was opened with an electric craniotomy saw, the brain was taken out and dissected in normal saline. Tissue collection was carried out separately from the 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 in both cerebral hemispheres. Each tissue sample was cut into two parts and frozen immediately on dry ice, and then stored at -80 ℃ for further evaluation (DNA and RNA extraction).

[0127] RNA extraction

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

[0129] Construction of cDNA library

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

[0131] Generation of tissue-specific libraries

[0132] Tissue-specific AAV libraries ("tissue libraries") were generated by PCR amplification of the region containing the 21 nt insert for deep sequencing.

[0133] Tissue enrichment score analysis

[0134] The enrichment score of each variant capsid polypeptide was calculated to evaluate its biodistribution in different tissues. In particular, each "enrichment score" was calculated using the following formula:

[0135]

[0136] Wherein the “ratio of the tissue library” is defined as the ratio of the reads of the AAV variants in the tissue library to the total reads of the tissue library; and the “ratio of the AAV library” is defined as the ratio of the reads of the AAV variants in the AAV library to the total reads of the AAV library.

[0137] After the first round of screening as described above, 8,162 variant capsid polypeptides with enrichment scores greater than 100 were selected from the collected tissues. Figure 2 The corresponding nucleotide sequences of the selected variants were synthesized according to steps 3-5 of the flowchart shown to generate a second round DNA library (R2 library) to further identify the best tissue-specific variants for each brain region.

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

[0139] As described above, the nucleotide sequences of 8,162 capsid variants selected from the first round of library screening were synthesized and used to construct a new plasmid library (Round 2 library; R2 library). The construction of the Round 2 library was identical to the Round 1 library as described earlier, and the library was injected into two cynomolgus monkeys (one female and one male).

[0140] In vivo screening and sequence analysis were performed with the round 2 AAV library following the same procedures as the round 1 library as described in Example 2. Deep sequencing analysis identified the number of insert sequences detected in each brain region as shown in Table 1. L indicates the left hemisphere; R indicates the right hemisphere.

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

[0142]

[0143] Among the variants identified from the second round of library screening, 60 capsid candidates were selected based on the following two criteria: (1) high enrichment score in at least one brain region (cutoff ≥ 1000); and (2) higher enrichment score compared to two reference capsids, AAV9 and PHP.eB capsid. 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 Figure 3). Some candidates (including ViVec-N020, ViVec-N021, ViVec-N023 and ViVec-N033) showed higher enrichment scores in multiple disease-related brain regions (e.g., frontal and temporal lobes for frontotemporal dementia or hippocampus for Alzheimer's disease). Some candidates (like ViVec-N005 and ViVec-N006) were particularly enriched in one brain region. The inserted 21 nucleotides and 7 amino acid sequences of these selected 60 capsid variant candidates are shown in Tables 2 and 3, respectively. The complete sequence of the Cap gene and protein can be determined by inserting a separate sequence into the position 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. Inserted 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 selected AAV capsid candidates

[0151] The 60 capsid candidates from the second round of library screening were constructed into a two-plasmid system for AAV packaging, which included a major plasmid and a helper RC plasmid.

[0152] Preparation of AAV GFP barcoded library

[0153] The 60 capsid variants selected from round 2 screening were used to construct the corresponding helper plasmids. AAV was packaged by using a two-plasmid system consisting of the primary plasmid pAAV-CMV-GFP-lib and the helper plasmid.

[0154] like Figure 4 As shown, a main plasmid was constructed to contain the packaged genome and two ITR elements flanking a construct containing a luciferase gene operably linked to a GFP gene via a T2A peptide under the control of a CMV promoter. The construct also contained a barcode downstream of the GFP gene.

[0155] A helper plasmid was constructed to contain the ampicillin resistance gene, AAV2-REP78, Ad5ψ and other elements involved in AAV capsid translation and assembly, and the Cap sequence encoding the AAV capsid protein.

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

[0157] Reagents quantity Helper plasmid 12ug S 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 connected to the backbone vector by infusion. After plasmid extraction, the AAV transgenic main plasmid pAAV-CMV-GFP-lib (10 ng) and pUC19 (8 μg) were co-transfected with the AAV auxiliary plasmid (30 μg) into HEK293T cells / 15 cm culture dish using lipofectamine LTX and plus reagent (Invitrogen, 94756).

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

[0160] In vivo studies in cynomolgus monkeys

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

[0162] The same tissue collection procedure as R1 and R2 library screening was performed. Genomic DNA (gDNA) was extracted from the collected tissue. According to the manufacturer's instructions, a portion (30 mg) of frozen tissue was used to extract gDNA by using EZNA MicroElute Genomic DNA Kit (Omega, D3096-02). The concentration and quality of gDNA were determined by Qubit 4 fluorometer (Invitrogen, Q33226).

[0163] Tissue enrichment scores for multiple AAV variants

[0164] The vector genome copy number for each variant was determined by deep sequencing of genomic samples processed from collected tissues, including the parietal lobe, frontal lobe, insula, occipital lobe, temporal lobe, hippocampus, striatum, midbrain, cerebellum, thalamus, hypothalamus, pons, medulla, and spinal cord in both brain hemispheres. Figure 5-Figure 18 Shown in.

[0165] like Figure 5-Figure 18 As shown, the enrichment score of each capsid candidate was normalized to that 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 were superior to AAV9 in at least one brain region (relative enrichment score >1).

[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 arranged from high to low.

[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, 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 arranged from high to low.

[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, 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 high to low.

[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, ViVec 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 high to low.

[0170] like Fig. 9As shown, in the insula, 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, 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 high to low.

[0171] like Fig.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 arranged from high to low.

[0172] like Fig.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, 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 high to low.

[0173] like Fig.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 high to low.

[0174] like Fig.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 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 Fig.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 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 arranged from high to low.

[0176] like Fig.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, 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 arranged from high to low.

[0177] like Fig.16As shown, in the medulla, 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, ViVec 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 arranged from high to low.

[0178] like Fig.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, 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 arranged from high to low.

[0179] like Fig.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, 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 arranged from high to low.

[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 dibutyryl-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 in Example 4, (2) AAV9 as a reference, or (3) AAV-PHP.eB as another reference at an MOI of 1E+4 vg / cell or 1E+5 vg / cell. Cultures were collected 72 h after transduction, RNA was extracted therefrom, and reverse transcribed into cDNA. AAV capsids in DA neurons were amplified, and the enrichment score of each AAV variant was determined by deep sequencing.

[0183] To identify rAAVs that displayed stronger transgene expression compared to AAV9 and thus had improved transduction efficiency, the fold change was calculated as the ratio of the enrichment score of rAAV to that of AAV9. Fig.19 The table in shows the log2 (fold change) values ​​of each rAAV calculated based on data obtained from repeated experiments. Values ​​of log2 (fold change) above 0 indicate higher expression levels. Fig.19As shown, it was found that 37 variants showed stronger transgenic GFP expression compared with wild-type AAV9. The 37 variants were 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, 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. Individual in vitro validation of selected AAV capsid candidates

[0185] Preparation of transgenic plasmids and helper plasmids

[0186] The main plasmid pAAV-CMV-Luc-T2A-GFP was constructed by inserting the construct CMV-Luc-T2A-GFP containing the gene of interest (GOI, in this case GFP and luciferase linked by the T2A peptide and under the control of the CMV promoter) into the plasmid px602 (Addgene). Fig. 20 As shown, the plasmid contains the following elements from 5' to 3': 5' ITR, CMV promoter, GOI (GFP and luciferase coding sequences linked by T2A) and poly A.

[0187] The RC helper plasmid contains AAV2-REP78, Ad5ψ, and other elements that aid in translation and assembly of the AAV capsid, as well as the Cap sequence encoding the heptamer of the AAV capsid protein inserted between amino acid residues 588 and 589.

[0188] Preparation of AAV variants for individual validation

[0189] Individual variant capsids containing rAAV were prepared to evaluate their ability to transduce human dopaminergic neurons. The main plasmid pAAV-CMV-Luc-T2A-GFP and the RC helper plasmid containing the individual capsid variant sequences were co-transfected into HEK293T cells using lipofectamine LTX and plus reagents. 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 genome titer was determined by qPCR. 20 μL of purified rAAV vector was treated 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 Master Mix and a primer / probe set that specifically binds to a sequence fragment of the luciferase gene.

[0192] The samples were diluted 10-fold (five concentrations ranging from 10 12 -10 8 Copies / mL), plasmid pAAV-CMV-Luc-T2A-GFP was used to prepare a standard curve for PCR quantification. In a 20 μL reaction mixture, the final concentrations of the reagents were: 0.4 μM primers targeting GFP, 0.4 μM probe, 1×ROX reference dye, 2 μL template, and 1× premix. PCR was run under the following conditions in a CFX96Touch real-time PCR detection system (Bio-Rad): 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 by 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, purified virus (2 × 10 10 The 1000 ng / ml particles were mixed with protein loading dye and heated at 95°C for 10 min.

[0195] The samples were then separated on SurePAGE 4%-12% Bis-Tris gel (GenScript, M00653). After electrophoresis, the gel was stained by Pierce Silver Staining Kit (Thermo Scientific, 24612) according to the manufacturer's instructions. In brief, the gel was washed twice with distilled water on a shaker for 5 min each time, then fixed in a fixing solution (volume ratio of water: anhydrous ethanol: acetic acid = 6: 3: 1) for 15 min and then fixed for 2 h. The gel was washed twice with 10% ethanol and water for 5 min each time. After incubation in 2‰ silver stain sensitizer for 1 min, the gel was quickly washed twice with water for 1 min each time. After the gel was soaked in silver stain for 30 min in the dark, a silver stain developer was added. Once the target band appears, 5% acetic acid is added to terminate the reaction, and the image is captured using a Bio-Rad gel imager.

[0196] Example 7. In vitro evaluation of selected AAV capsid candidates in primary neuronal cells

[0197] The first 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 for Rep and Cap (RC helper plasmids), generating RC-001, RC-002, RC-004, and RC-023. Each specific RC helper plasmid containing Cap was packaged with the pAAV-CMV-Luc-T2A-GFP plasmid to generate capsid-specific AAV for further evaluation.

[0198] Human DAD25 and DAD45 were maintained in culture medium as 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+4vg / cell or 1E+5vg / cell. AAV9 and PHP.eB were used as controls. 72 hours after infection, luciferase levels were measured using the Bright-Lite luciferase assay, and luminescence intensity was analyzed using a microplate analyzer. The transduction efficiency of the individual AAV variants was determined by comparing the luminescence intensity with that of wild-type AAV9.

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

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

[0201] For testing in cortical neurons, AAV capsid variants were prepared by co-transfection of the AAV transgene master plasmid pAAV-CMV-Luc-T2A-GFP (8 μg), RC plasmid (30 μg) and AAV helper plasmid (30 μg) into HEK293T cells / 15 cm dish using a three-plasmid system with lipofectamine LTX and plus reagent. Transfected cells and culture medium were harvested 72 h later and subsequently centrifuged and purified 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 the feed medium at MOI=1E+4 and 1E+5. Five days after AAV transduction, cells were harvested, and the infection efficiency of each variant was determined by luciferase assay as described above. The results show that in human cortical neurons, ViVec-N001 and ViVec-N002 are superior to AAV9.

[0203] The transduction efficiency of the AAV variants was also evaluated in immature iPSC-derived human motor neurons (prepared from human motor progenitor cells, HopStem#) 21 days after differentiation. Fig.23 As shown, both ViVec-N001 and ViVec-N002 performed better than AAV9.

[0204] In conclusion, ViVec-N002 showed the best transduction efficiency in these neuronal subtypes except DA neurons, indicating its potential to efficiently deliver GOIs to different regions of the nervous system for the treatment of various neuronal diseases.

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

[0206] Example 8. Individual in vivo validation of selected AAV capsid candidates

[0207] Several capsid candidates were selected for further evaluation of their in vivo biodistribution in the central nervous system.

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

[0209] Compared with AAV9, both ViVec-N001 and ViVec-N004 showed stronger GFP expression in the striatum ( Fig.24A In the hippocampus, ViVec-N001 was superior to AAV9 ( Fig. 24B In the medulla, ViVec-N001 and ViVec-N004 showed GFP expression levels comparable to AAV9 ( Fig.24C ). Given that the medulla is the area closest to the injection site, the above observations could indicate that both variants, ViVec-N001 and ViVec-N004, and especially ViVec-N001, could diffuse to more distant areas and most likely provide a wider distribution in the nervous system.

[0210] Candidates were further evaluated by different routes of administration. ViVec-N002, ViVec-N033 and ViVec-N022 (2.5e12 per animal) carrying luciferase and GFP reporter genes were delivered to the brain of cynomolgus monkeys via intracerebroventricular injection. 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 luciferase expression were determined by qPCR (at the DNA and mRNA levels) and immunostaining (at the protein level). The results are in Figure 25-27 Shown in.

[0211] Overall, ViVec-N002, ViVec-N033, and ViVec-N022 showed broader and more robust biodistribution than AAV9. Fig.25ViVec-N002 was shown to achieve higher transduction efficiency than AAV9 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 ganglia, and thoracic dorsal root ganglia. Based on earlier data, ViVec-N033 and ViVec-N022 are expected to perform similarly to ViVec-N002.

[0212] like Fig.26 As shown, ViVec-N022 showed higher levels of luciferase DNA (shown as relative fold change relative to AAV9) in the following areas: occipital cortex (lobe), temporal cortex (lobe), medulla, striatum, midbrain, pons, spinal cord-thoracic, spinal cord-lumbar, olfactory bulb, and optic nerve, and much less in peripheral organs and dorsal root ganglia (DRG). ViVec-N033 showed a partially different distribution pattern, with relatively higher levels of luciferase DNA in the following areas: 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 gland. Similar to ViVec-N022, ViVec-N033 also had lower distribution in peripheral organs and DRG.

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

[0214] The above candidates were further evaluated by intrastriatal administration. ViVec-N001 and ViVec-N002 carrying the GFP reporter gene were delivered into the mouse striatum by intrastriatal injection (2e12 vg per injection site in the striatum). Fig.28As shown, the striatum region of ViVec-N002 transduction is more extensive than the striatum region of AAV9 transduction.ViVec-N033 and ViVec-N022 are also tested by intrastriatal administration route.By intrastriatal injection, 1.2E+10vg of ViVec-N033 and ViVec-N022 are injected into mouse striatum respectively, 21 days after injection, striatal tissue is collected, and RT-qPCR is used to determine the RNA level of luciferase.We found that compared with AAV9, ViVec-N033 and ViVec-N022 both delivered more luciferase (GOI), which is similar to ViVec-N002.

[0215] The ability of individual candidates to deliver genes into the central nervous system via intrathecal administration was also evaluated. ViVec-N033 and ViVec-N022 carrying both luciferase and GFP reporter genes were injected into SD rats via intrathecal injection (2 doses, low: 1E+13vg / kg and high: 3E+13vg / kg). AAV9 was used as a reference. 21 days after injection, the RNA levels of luciferase in spinal cord and cortical tissues were determined by RT-qPCR. Fig.30 As shown, both ViVec-N033 and ViVec-N022 had better transfection efficiency than AAV9 and expressed more luciferase RNA in the thoracic spinal cord, lumbar spinal cord (injection site) and cortex.

[0216] The candidate was further evaluated by intravenous administration route to test the ability of these novel capsids to pass through the blood-brain barrier. Each of ViVec-N002, ViVec-N033 and ViVec-N022 carrying luciferase and GFP reporter genes was delivered to two different strains of mice via intravenous injection. 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 (on DNA and mRNA levels) and immunostaining (on protein levels).

[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 comprising an insertion of 7 amino acids at a position after amino acid position 588 of the capsid polypeptide of AAV9, The amino acid sequence of the AAV9 capsid polypeptide is shown in SEQ ID NO:

121. The engineered AAV capsid polypeptides are capable of assembling into recombinant AAV (rAAV) virions, and the rAAV virions have increased tropism for one or more tissues or cells of the central nervous system (CNS) and / or are capable of producing higher levels of transgene expression in tissues or cells of the CNS compared to rAAV virions having a capsid of serotype AAV9.

2. An engineered adeno-associated virus (AAV) capsid polypeptide, comprising an insertion of any amino acid sequence as shown in SEQ ID NO: 1-60 after amino acid position 588 of the capsid polypeptide of AAV9.

3. An engineered adeno-associated virus (AAV) capsid polypeptide according to claim 1 or claim 2, wherein the rAAV virion has increased tropism for one or more tissues or cells of the CNS and / or is capable of producing higher levels of transgene expression in tissues or cells of the CNS compared to rAAV virions having a capsid of serotype AAV9, wherein the tissues or cells are selected from the parietal lobe, frontal lobe, temporal lobe, occipital lobe, insula, striatum, hippocampus, midbrain, thalamus, hypothalamus, cerebellum, medulla, pons, spinal cord, cortical neurons, motor neurons, dopaminergic neurons, astrocytes or oligodendrocytes.

4. The engineered adeno-associated virus (AAV) capsid polypeptide of any one of claims 1-3, wherein the rAAV virion has increased tropism for one or more tissues or cells of the CNS and / or is capable of producing higher levels of transgene expression in tissues or cells of the CNS compared to rAAV virions having a capsid of serotype AAV-PHP.eb.

5. A recombinant adeno-associated virus (rAAV) virion, comprising a capsid assembled from the AAV capsid polypeptide according to claim 1 and a vector genome containing a transgene.

6. The rAAV virion of claim 5, wherein the transgene is a therapeutic gene or a reporter gene.

7. A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide according to any one of claims 1 to 4.

8. The nucleic acid molecule according to claim 6, comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 61-120.

9. A recombinant adeno-associated virus (rAAV) helper vector, comprising a nucleotide sequence encoding a polypeptide according to any one of claims 1-4.

10. A pharmaceutical composition comprising the rAAV virus particles according to claim 5 or claim 6 and a pharmaceutically acceptable carrier.

11. Use of the rAAV virus particle according to claim 5 or claim 6 or the pharmaceutical composition according to claim 10 in the manufacture of a medicament.

12. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the rAAV virus particle according to claim 5 or claim 6 or the pharmaceutical composition according to claim 10.

13. An rAAV library comprising a plurality of variant AAV capsid polypeptides, each of the AAV capsid polypeptides having an insertion of 7 amino acids 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 is randomly selected from any one of 20 amino acids.

14. The rAAV library according to claim 13, wherein the AAV of the AAV library has at least 1×10 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 Types of variant capsid polypeptides.

15. A DNA or plasmid library comprising a plurality of nucleotide sequences encoding variant AAV capsid polypeptides, each of which has an insertion of 7 amino acids between amino acid position Q588 and amino acid position A589 of the wild-type AAV9VP1 capsid protein as shown in SEQ ID NO:

121.

16. The DNA or plasmid library according to claim 15, encoding at least 1×10 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 Types of variant capsid polypeptides.

17. Use of the library according to any one of claims 13 to 16 for screening rAAVs having desired properties.

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