AAV capsids and vectors for transduction of cells

Abstract

The present disclosure relates generally to adeno-associated virus (AAV) capsid polypeptides and encoding nucleic acid molecules. The disclosure also relates to AAV vectors comprising the capsid polypeptides, and nucleic acid vectors (e.g. plasmids) comprising the encoding nucleic acids molecules, as well as to host cells comprising the vectors. The disclosure also relates to methods and uses of the polypeptides, encoding nucleic acids molecules, vectors and host cells.

Description

AAV capsids and vectors for transduction of cellsRelated Applications

[0001] This application claims priority to Australian Provisional Application No. 2024900077 entitled "AAV capsids and vectors for transduction of cells" filed 11 January 2024, the contents of which are incorporated herein by reference in their entirety.Field of the Disclosure

[0002] The present disclosure relates generally to adeno-associated virus (AAV) capsid polypeptides and encoding nucleic acid molecules. The disclosure also relates to AAV vectors comprising the capsid polypeptides, and nucleic acid vectors e.g. plasmids) comprising the encoding nucleic acids molecules, as well as to host cells comprising the vectors. The disclosure also relates to methods and uses of the polypeptides, encoding nucleic acids molecules, vectors and host cells.Background of the Disclosure

[0003] Gene therapy has most commonly been investigated and achieved using viral vectors, with notable recent advances being based on adeno-associated viral vectors. Adeno- associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length. The AAV genome includes inverted terminal repeat (ITRs) at both ends of the molecule, flanking two open reading frames: rep and cap. The cap gene encodes three capsid proteins: VP1, VP2 and VP3. The three capsid proteins typically assemble in an estimated ratio of 1: 1: 10 to form the AAV capsid, although AAV capsids containing only VP3, or VP1 and VP3, or VP2 and VP3, have been produced. The cap gene also encodes the assembly activating protein (AAP) from an alternative open reading frame. AAP promotes capsid assembly, acting to target the capsid proteins to the nucleolus and promote capsid formation. The rep gene encodes four regulatory proteins: Rep78, Rep68, Rep52 and Rep40. These Rep proteins are involved in AAV genome replication.

[0004] The ITRs are involved in several functions, in particular integration of the AAV DNA into the host cell genome, as well as genome replication and packaging. When AAV infects a host cell, the viral genome can integrate into the host's chromosomal DNA resulting in latent infection of the cell. Thus, AAV can be exploited to introduce heterologous sequences into cells. In nature, a helper virus (for example, adenovirus or herpesvirus) provides protein factors that allow for replication of AAV virus in the infected cell and packaging of new virions. In the case of adenovirus, genes E1A, E1B, E2A, E4 and VA provide helper functions. Upon infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and the helper virus are produced.

[0005] AAV vectors (also referred to as recombinant AAV, rAAV) that contain a genome that lacks some, most or all of the native AAV genome and instead contains one or more heterologoussequences flanked by the ITRs have been successfully used in gene therapy settings. These AAV vectors are widely used to deliver heterologous nucleic acid to cells of a subject for therapeutic purposes. Recombinant AAV vectors are increasingly showing therapeutic promise in targeting organs and cells that necessitate direct in vivo gene delivery.

[0006] An ongoing challenge is targeted and specific delivery to the desired cells with reduced or negligible delivery to other cells in the body. For example, AAV-mediated therapy of central nervous system (CNS) disorders is most desirably performed using systemic administration (e.g. intravenous (IV) administration), or alternatively intra-cerebrospinal fluid (CSF) administration (e.g. intrathecal (IT) administration), requiring AAV capsids to efficiently cross the blood-brain barrier (BBB). However, this often results in significant transduction of off-target tissues in the periphery, with high risk of toxicity, particularly at high vector doses. For example, significant side effects have been observed with the market approved gene therapeutic, Zolgensma™, which uses AAV9 to deliver a functional copy of the SMN1 gene for the treatment of spinal muscular atrophy (SMA). Although Zolgensma™ has had a revolutionary impact on lives of SMA patients and their families, liver toxicity in patients has raised concerns about the safety of AAV9-based gene therapies, as well as rAAV-based therapies requiring systemic delivery in general (Feldman et al. J Pediatr. 2020;225:252-258; Chand et al. J Hepatol. 2021;74(3):560-566). There remains a need, therefore, for AAV vectors with reduced off-target transduction of cells in, for example, the liver.Summary of the Disclosure

[0007] The present disclosure is predicated in part on the identification of novel AAV capsid polypeptides. Typically, the capsid polypeptides, when present in the capsid of an AAV vector, facilitate transduction of cells, such as transduction of cells in the CNS, while avoiding or mostly avoiding transduction of liver cells.

[0008] In particular, and as demonstrated herein, the AAV vectors comprising capsid polypeptides of the present disclosure can exhibit de-targeting from both mouse and human hepatocytes when compared to clinically relevant AAV9, both in vitro and in vivo. The identified new variants provided herein also exhibit substantial improvement over AAV9 for delivering transgenes to the CNS via i.v. injection. The data provided herein demonstrates that these AAV capsid variants not only surpass AAV9 in terms of transduction efficiency at the DNA (cell entry) and RNA (transgene expression) level in the CNS, but also demonstrate clear neuronal tropism and regional bias for the brain stem and mid-brain structures, and liver de-targeting. As a result of these properties, the AAV vectors comprising the capsids have improved and desirable safety and CNS targeting profiles, such as following IV or IT administration, with reduced off-target effects in the liver compared to AAV9. Additionally, as also demonstrated herein, the AAV capsids may optionally be engineered for immune evasion, resulting in improved immune escape properties beyond those of AAV9. This enhancement in immune escape mechanisms is crucial for evading immune responses and maximising the therapeutic potential of the gene therapy approach.

[0009] Thus, in one aspect, provided is an AAV capsid polypeptide, wherein the capsid polypeptide: a) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3-13; b) comprises an amino acid sequence having at least 96%, 97%, 98% or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 3-13; c) comprises an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to the sequence set forth in any one of SEQ ID NOs: 3-13 and comprises one or more modifications selected from among D4G, Y6C, N14T, E21Q, D24A, D24K, A29P, A29V, K31Q, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, D41N, G42S, G42R, F56G, A67E, R92K, Q105K, V125L, L129F, G135A, G141A, Q148P, 151- 152insR, E152S, S157T, T162K, K168R, D178E, S179T, P185D, L188I, S205A, A263Q, T265del, S268del, T326Q, V330T, S345T, I373V, I373N, N383D, T411Q, T415E, E418N, E418D, N497Q, N498del, S499Q, N500Q, F501L, T502A, F584L, A598V, A601V, H628N, L640M, N642H, V699I, A706Y, A709T, N710S, T714A, D716N, N717T, N718E, L720V, T722S and P735N; or d) is a VP2 or VP3 fragment of any one of a) - c), wherein the VP2 fragment comprises residues at the positions corresponding to positions 138-736 of the AAV1 polypeptide set forth in SEQ ID NO: 1 and wherein the VP3 fragment comprises residues at the positions corresponding to positions 204-736 of the AAV1 polypeptide set forth in SEQ ID NO: 1.

[0010] In some examples, the AAV capsid polypeptide comprises one or more modifications selected from among D24A, A29V, K31Q, K38H, D41N, G42R, L129F, G135A, G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, D178E, P185D, S205A, S268del, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N497Q, N498del, S499Q, N500Q, F501L, T502A, F584L, A598V, A601V, H628N, L640M, N642H and V699I. In particular examples, the polypeptide comprises the mutation N498del and / or the mutation T502A relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

[0011] In a further aspect, provided is an AAV capsid polypeptide, wherein the capsid polypeptide comprises an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity to the sequence set forth in any one of SEQ ID NOs: 3-9 or 11-13, or a VP2 or VP3 fragment thereof, wherein: the VP2 fragment comprises residues at the positions corresponding to positions 138- 736 of the AAV1 polypeptide set forth in SEQ ID NO: 1 and wherein the VP3 fragment comprises residues at the positions corresponding to positions 204-736 of the AAV1 polypeptide set forth in SEQ ID NO: 1; and the capsid polypeptide comprises the modifications N498del and T502A relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

[0012] In some examples, the AAV capsid polypeptide further comprises (in addition to modifications N498del and T502A) one or more modifications selected from among D4G, Y6C, N14T, E21Q, D24A, D24K, A29P, A29V, K31Q, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, D41N, G42S, G42R, F56G, A67E, R92K, Q105K, V125L, L129F, G135A, G141A, Q148P, 151- 152insR, E152S, S157T, T162K, K168R, D178E, S179T, P185D, L188I, S205A, A263Q, T265del, S268del, T326Q, V330T, S345T, I373V, I373N, N383D, T411Q, T415E, E418N, E418D, N497Q, N498del, S499Q, N500Q, F501L, T502A, F584L, A598V, A601V, H628N, L640M, N642H, V699I, A706Y, A709T, N710S, T714A, D716N, N717T, N718E, L720V, T722S and P735N ; such as one or more modifications selected from among D24A, A29V, K31Q, K38H, D41N, G42R, L129F, G135A,G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, D178E, P185D, S205A, S268del, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N497Q, S499Q, N500Q, F501L, F584L, A598V, A601V, H628N, L640M, N642H and V699I relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

[0013] In one example, the AAV capsid polypeptide comprises N498del and T502A and one or more modifications selected from among T326Q, V330T, S345T, N383D, T411Q, T415E and E418N relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1. In a particular example, the polypeptide comprises modifications T326Q, V330T, S345T, N383D, T411Q, T415E and E418N relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

[0014] In another example, the AAV capsid polypeptide comprises N498del and T502A and one or more modifications selected from among from D24A, K31Q and D178E relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1. In a particular example, the AAV capsid polypeptide comprises modifications D24A, K31Q, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del and T502A, relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

[0015] In another example, the AAV capsid polypeptide comprises N498del and T502A and one or more modifications selected from G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, P185D, S205A, N642H and V699I relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1. In a particular example, the AAV capsid polypeptide comprises modifications G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, P185D, S205A, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, 642H and V699I relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

[0016] In another example, the AAV capsid polypeptide comprises N498del and T502A and one or more modifications selected from D24A, G135A, Q148P, 151-152insR, E152S, S157T, T162K, D178E, A598V, A601V, H628N, L640M and N642H relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1. In a particular example, the AAV capsid polypeptide comprises modifications D24A, G135A, Q148P, 151-152insR, E152S, S157T, T162K, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, A598V, A601V, H628N, L640M and N642H relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

[0017] In another example, the AAV capsid polypeptide comprises N498del and T502A and one or more modifications selected from D24A, A29V, K31Q, K38H, D41N, G42R, L129F, Q148P, 151- 152insR, E152S, S157T, T162K, D178E, N497Q, S499Q, N500Q, F501L, F584L, A598V and N642H relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1. In a particular example, the AAV capsid polypeptide comprises modifications D24A, A29V, K31Q, K38H, D41N, G42R, L129F, Q148P, 151-152insR, E152S, S157T, T162K, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N497Q,N498del, S499Q, N500Q, F501L, T502A, F584L, A598V and N642H relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

[0018] In another example, the AAV capsid polypeptide comprises N498del and T502A and one or more modifications selected from D24A, Q148P, 151-152insR, E152S, S157T, T162K, D178E,S268del, F584L and A598V relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1. In a particular example, the AAV capsid polypeptide comprises modifications D24A, Q148P, 151- 152insR, E152S, S157T, T162K, D178E, S268del, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, F584L and A598V relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

[0019] In some examples, the AAV capsid polypeptide also comprises a modification that confers increased immune evasion on an AAV vector comprising the capsid polypeptide, e.g. a replacement of 456-AQNK-459 with 456-SERR-459; or replacement of 588-STDPATGDVH-597 with 588-DLDPKATEVE-597.

[0020] Also provided is an AAV vector, comprising the capsid polypeptide described above and herein.

[0021] In some examples, the AAV vector exhibits decreased transduction efficiency of a cell of the liver, heart, kidney or muscle compared to an AAV9 vector comprising a capsid polypeptide comprising the amino acid sequence of SEQ ID NO:2. In particular examples, the vector exhibits decreased transduction efficiency of a hepatocyte compared to an AAV1 or AAV9 vector comprising a capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2. In some examples, transduction efficiency is decreased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0022] In one example, the AAV vector exhibits increased in vivo transduction efficiency of a cell of the central nervous system compared to an AAV1 or AAV9 vector comprising a capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2. In particular examples, the cell of the central nervous system is in the brainstem, cerebellum, midbrain, thalamus, spinal cord, cortex or hippocampus, and / or transduction efficiency is increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or 500%.

[0023] The AAV vector may further comprises a heterologous coding sequence, e.g. one that encodes a peptide, polypeptide or polynucleotide, such as a therapeutic peptide, polypeptide or polynucleotide.

[0024] Also provided is an isolated nucleic acid molecule encoding a capsid polypeptide described above and herein, and a vector comprising the nucleic acid molecule. In some examples, the vector is selected from among a plasmid, cosmid, phage and transposon.

[0025] Also provided is a host cell, comprising a AAV vector, a nucleic acid molecule, or a vector described above and herein.

[0026] Also provided is a method for introducing a heterologous coding sequence into a host cell (e.g. a cell of the central nervous system) comprising contacting a host cell with the AAV vector. In some examples, contacting the host cell with the AAV vector comprises administering the AAV vector to a subject. Administration of the AAV vector to the subject may effect treatment of adisease or condition of the central nervous system. In other examples, the method is performed in vitro or ex vivo.

[0027] Also provided is use of the AAV vector for the preparation of a medicament for treating a disease or condition of the central nervous system.

[0028] Also provided is a method for producing an AAV vector, comprising culturing a host cell comprising a nucleic acid molecule encoding a capsid polypeptide described above or herein, an AAV rep gene, a heterologous coding sequence flanked by AAV inverted terminal repeats, and helper functions for generating a productive AAV infection, under conditions suitable to facilitate assembly of an AAV vector comprising a capsid comprising the capsid polypeptide, wherein the capsid encapsidates the heterologous coding sequence.Brief Description of the Drawings

[0029] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the following drawings.

[0030] Figure 1 - AAV transduction of FRG mice. Eleven 11 AAV-BBB variants were mixed at an equimolar ratio along with AAV9 and injected i.v. in two males FRG mice at a total dose of 1.8 x 1011 total vg. Twenty-eight days post-injection, brain and spinal cord of the mice were harvested and sent for NGS analysis at the DNA level (representing physical transduction or cell entry) and cDNA level (representing functional transduction or protein expression) to determine the top performing variants. (A) DNA reads in the brain. (B) DNA reads in the spinal cord. (C) cDNA reads in the brain. (D) cDNA reads in the spinal cord. Data is represented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical significance was calculated using a one-way ANOVA with Dunnetts's multiple comparisons test with AAV9 as the control.

[0031] Figure 2 - AAV capsid alignments. Alignment of amino acid residues between AAV1, AAV- BBB3, AAV-BBB6, AAV-BBB28, AAV-BBB31 and AAV-BBB52. Amino acid residues in bold are those that differ from AAV1.

[0032] Figure 3 - CNS transduction profile of AAV-BBB variants compared with AAV9 and AAV1. Individual comparison of top performing AAV-BBB variants to AAV9. The capsid variants AAV9, AAV1, AAV-BBB6, AAV-BBB28 and AAV-BBB31 were packaged with a transgene containing a mCherry reporter under the control of the ubiquitous CMV promoter. Each individual capsid variant was i.v. administered into male FRG mice (N = 3, dose: 5 x 1011total vg / animal). Mouse tissues were harvested three weeks post injection. Vector copy number (VCN) was assessed, represented by vg per diploid cell (normalized to mouse GAPDH). (A) VCN in the brain. (B) VCN in the spinal cord. (C) VCN in the liver and represented by vg per diploid cell (normalized to mouse GAPDH). Data is represented as the mean ± SEM. *p < 0.05, **p < 0.01. Statistical significance was calculated using a one-way ANOVA with Dunnetts's multiple comparisons test with AAV9 and AAV1 as the controls.

[0033] Figure 4 - Neuronal transduction of AAV-BBB variants. Following individual injection of the AAV-BBB variants and AAV9 i.v. in FRG mice (N = 3, dose: 5 x 1011total vg / animal), tissues were harvested three weeks post injection for down-stream analysis. (A) Percentage of mCherry positive neurons in the brainstem, cerebellum, cortex, midbrain, hippocampus, and thalamus of mice injected with AAV9 or AAV-BBB variants. Data is represented as the mean ± SEM. Individual data points represent the average of 3-5 non-overlapping images per region taken with a 20x objective. **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical significance was calculated using a two-way ANOVA with Dunnetts's multiple comparisons test with AAV9 as the control. (B) Percentage of mCherry positive neurons in the spinal cord of mice injected with AAV9 or AAV-BBB variants. Data is represented as the mean ± SEM. *p < 0.05, **p < 0.01. Statistical significance was calculated using a one-way ANOVA with Dunnetts's multiple comparisons test with AAV9 as the control.

[0034] Figure 5 - Utilisation of the hSyn promoter with AAV-BBB variants. Capsid variants AAV9, AAV-BBB28 and AAV-BBB31 were packaged with a transgene containing an mCherry reporter under the control of the hSyn promoter. Each individual capsid variant was administered i.v. to male FRG mice with a dose of 5 x 1011vg / animal. Transgene expression was assessed by mCherry fluorescence three weeks post injection. Quantification of the number of mCherry positive neurons in the cortex. Data is represented as the mean ± SEM. Individual data points represent the average of 3-4 non-overlapping images of the cortex region. ***p < 0.001. Statistical significance was calculated using a two-way ANOVA with Sidak's multiple comparisons test.

[0035] Figure 6 - Liver-detargeting of AAV-BBB variants. Individual comparison of topperforming AAV-BBB variants to AAV9 and AAV1. The capsid variants AAV9, AAV-BBB28 and AAV- BBB31 were packaged with a transgene containing a GFP reporter under the control of the ubiquitous CMV promoter. Each individual capsid variant was intravenously administered into N = 2 humanised FRG (hFRG) mice (dose: 5 x 1011total vg / animal). Mice tissues were harvested four weeks post injection, with one lobe of the chimeric mouse / human liver collected for IHC analysis. The remaining liver was used to isolate the human hepatocyte population using FACS for analysis of the VCN. (A) VCN analysis of human hepatocytes isolated using FACS. (B) Percentage GFP positive cells per human cluster (N = 12-15 human clusters per mouse were analysed). Data is represented as the mean ± SEM. *p < 0.05, ****p < 0.0001. Statistical significance was calculated using a one-way ANOVA with Dunnetts's multiple comparisons test with AAV9 as the control.

[0036] Figure 7 - CNS Transduction Profile of AAV-BBB28 and AAV-BBB31 in hFRG mouse CNS as compared with AAV9. Capsids were packaged with an eGFP reporter driven by a ubiquitous CMV promoter. hFRG mice were injected intravenously at a dose of 5 x 1011vg / animal and harvested 3 weeks post injection. (A) VCN of each variant in the brain. (B) VCN of each variant in the brain spinal cord. Data is represented as the mean ± SEM. **p < 0.01. Statistical significance was calculated using a one-way ANOVA with Dunnetts's multiple comparisons test against AAV9 as the control.

[0037] Figure 8 - Assessment of AAV-BBB escape variants. (A-C) Neutralisation assay of indicated AAV vectors following pre-incubation with human IVIg prior to transduction of HEK293T cells. The percentage of GFP-positive cells 48h after transduction was analysed by flow cytometry (N = 3 independent experiments). The dotted line represents IVIg-mediated inhibition of AAV transduction by 50%. (D-E) CNS transduction profile of AAV-BBB28.VR-IV (N = 3) and AAV- BBB28.VR-VIII (N = 2) in FRG mouse CNS as compared with parental AAV-BBB28 (N = 2). 8- weeks old FRG mice were injected i.v. with a dose of 2 x 1011total vg / animal. Mice were harvested 3 weeks post injection for IHC and VCN analysis. (D) VCN of each variant in the brain. (E) VCN of each variant in the spinal cord. Data is represented as the mean ± SEM.

[0038] Figure 9 - Assessment of AAV-BBB6 variants. (A) Neutralisation assay of indicated AAV- BBB6 variants following pre-incubation with human IVIg prior to transduction of HEK293T cells. The percentage of GFP-positive cells 48 h after transduction was analysed by flow cytometry (N = 3 independent experiments). The dotted blue line represents IVIg-mediated inhibition of AAV transduction by 50% (IC50). (B)-(C) CNS Transduction profile of AAV-BBB6.VR-IV and AAV- BBB6.VR-VIII in FRG mouse CNS as compared with wtAAV-BBB6. (B) VCN of each variant in the brain. (C) VCN of each variant in the spinal cord.

[0039] Figure 10 - Brain transduction of AAV-BBB variants in BALB / cJ mice. AAV vector performance following i.v. injection of AAV-BBB28 or AAV-BBB31 compared to AAV9 was assessed in BALB / cJ mice. All variants were used to package a transgene cassette encoding an mCherry reporter under the control of the ubiquitous CMV promoter and injected at a dose of 5 x 1011total vg / animal. (A) VCN analysis of the BALB / cJ mouse brains following i.v. injection. VCN was determined by ddPCR of the viral genomes. Data is shown as mean ± SEM. N = 3. ****p < 0.0001. Statistical significance was calculated using a one-way ANOVA with Dunnetts's multiple comparisons test with AAV9 as the control. (B) Cell binding assay of AAV9, AAV-PHP.eB, AAV- BBB28 and AAV-BBB31 in HEK293T transfected with Ly6a or untransfected control. A MOT of 10,000 vg / cell was used. Binding was assessed by ddPCR of the viral genome. Data is shown as mean ± SEM. N = 3. (C) Transduction assay of aforementioned AAV variants as measured by FACS 48 hours post transduction (percentage of GFP positive cells). (D) Transduction assay of aforementioned AAV variants as measured by FACS 48 hours post transduction (mean fluorescence intensity (MFI) quantification). Data is shown as mean ± SEM. N = 3. For all in vitro analysis,. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical significance was calculated using a two-way ANOVA with Sidak's multiple comparisons test.

[0040] Figure 11 - In vivo comparison of AAV1, AAV-BBB6, and the indicated swapped variants in the brain of non-engrafted FRG mice (N = 2) as assessed by NGS at the DNA level. Percentage of NGS reads are mapped to each barcoded transgene for individual assessment (n = 3 barcodes / capsid). (A) C1-C6 variants in the brain (mouse #137 and #138). (B) AAV1-N498del and AAV1-T502A variants in the brain (mouse #242 and #250). (C) AAV1-N498del, AAV1-T502A, AAV-BBB6-498insN and AAV-BBB6-A501T variants in the brain (mouse #251 and #252).

[0041] Figure 12 - In vivo comparison of AAV1, AAV-BBB6, and the indicated swapped variants in the brain of non-engrafted FRG mice (N = 2) as assessed by NGS at the DNA level. AAV1, AAV- BBB6, AAV1-T502A, AAV1-N498del, AAV1-N498del-T5O2A, and AAV-BBB6-A501T, AAV-BBB6- 498insN and AAV1RX. Each individual capsid variant was i.v. administered into male FRG mice (N = 2, dose: 5 x 1011total vg / animal). VCN was assessed in the brain and represented by vg per diploid cell (normalized to mouse actin). Data is represented as the mean ± SEM.

[0042] Figure 13 - In vivo comparison of 498N insertion and A501T mutation in other BBB variants (A) VP1 pairwise sequence alignment of AAV-BBB variants. Region surrounding the N498del and T501A positions is displayed. (B-C) In vivo barcoded NGS comparison of indicated variants in a non-engrafted FRG mice (N = 2, dose: 1 x 1012 total vg / animal). (B) Percentage of NGS reads mapped to each AAV variant (n = 3 barcodes / capsid) in the brain at the DNA (cell entry, physical transduction) and cDNA (expression, functional transduction) level, normalized to the pre-injection mix. (C) Percentage of NGS reads mapped to each AAV variant (n = 3 barcodes / capsid) in the NeuN following nuclei isolation of NeuN positive nuclei via FACS at the DNA (cell entry, physical transduction) level, for Low and High NeuN population, normalized to the pre-injection mix.

[0043] Some figures and text contain color representations or entities. Color illustrations are available from the Applicant upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.Detailed Description

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the disclosure belongs. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information.

[0045] As used herein, the singular forms "a", "an" and "the" also include plural aspects ( / .e. at least one or more than one) unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a single polypeptide, as well as two or more polypeptides.

[0046] In the context of this specification, the term "about," is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[0047] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will beunderstood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0048] As used herein, a "vector" includes reference to both polynucleotide vectors and viral vectors, each of which are capable of delivering a transgene contained within the vector into a host cell. Vectors can be episomal, i.e., do not integrate into the genome of a host cell, or can integrate into the host cell genome. The vectors may also be replication competent or replication deficient. Exemplary polynucleotide vectors include, but are not limited to, plasmids, cosmids and transposons. Exemplary viral vectors include, for example, AAV, lentiviral, retroviral, adenoviral, herpes viral and hepatitis viral vectors.

[0049] As used herein, "adeno-associated viral vector" or "AAV vector" refers to a vector in which the capsid is derived from an adeno-associated virus, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, AAV from other clades or isolates, or is derived from synthetic, bioengineered or modified AAV capsid proteins, including chimeric capsid proteins. In particular embodiments, the AAV vector has a capsid comprising a capsid polypeptide of the present disclosure. When referring to AAV vectors, both the source of the genome and the source of the capsid can be identified, where the source of the genome is the first number designated and the source of the capsid is the second number designated. Thus, for example, a vector in which both the capsid and genome are derived from AAV2 is more accurately referred to as AAV2 / 2. A vector with an AAV6-derived capsid and an AAV2-derived genome is most accurately referred to as AAV2 / 6. A vector with the bioengineered DJ capsid and an AAV2-derived genome is most accurately referred to as AAV2 / DJ. For simplicity, and because most vectors use an AAV2-derived genome, it is understood that reference to an AAV6 vector generally refers to an AAV2 / 6 vector, reference to an AAV2 vector generally refers to an AAV2 / 2 vector, etc. An AAV vector may also be referred to herein as "recombinant AAV", "rAAV", "recombinant AAV virion", "rAAV virion", "AAV variant", "recombinant AAV variant", and "rAAV variant" terms which are used interchangeably and refer to a replication-defective virus that includes an AAV capsid shell encapsidating an AAV genome. The AAV vector genome (also referred to as vector genome, recombinant AAV genome or rAAV genome) comprises a transgene flanked on both sides by functional AAV ITRs. Typically, one or more of the wild-type AAV genes have been deleted from the genome in whole or part, preferably the rep and / or cap genes. Functional ITR sequences are necessary for the rescue, replication and packaging of the vector genome into the rAAV virion.

[0050] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chainsis lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying its activity.

[0051] The term "ITR" refers to an inverted terminal repeat at either end of the AAV genome. This sequence can form hairpin structures and is involved in AAV DNA replication and rescue, or excision, from prokaryotic plasmids. ITRs for use in the present disclosure need not be the wildtype nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, as long as the sequences provide for functional rescue, replication and packaging of rAAV.

[0052] As used herein, "functional" with reference to a capsid polypeptide means that the polypeptide can self-assemble or assemble with different capsid polypeptides to produce the proteinaceous shell (capsid) of an AAV virion. It is to be understood that not all capsid polypeptides in a given host cell assemble into AAV capsids. Preferably, at least 25%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95% of all AAV capsid polypeptide molecules assemble into AAV capsids. Suitable assays for measuring this biological activity are described e.g. in Smith-Arica and Bartlett (2001), Curr Cardiol Rep 3(1): 43-49.

[0053] "AAV helper functions" or "helper functions" refer to functions that allow AAV to be replicated and packaged by a host cell. AAV helper functions can be provided in any of a number of forms, including, but not limited to, as a helper virus or as helper virus genes which aid in AAV replication and packaging. Helper virus genes include, but are not limited to, adenoviral helper genes such as E1A, E1B, E2A, E4 and VA. Helper viruses include, but are not limited to, adenoviruses, herpesviruses, poxviruses such as vaccinia, and baculovirus. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C (Ad5) is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and are available from depositories such as the ATCC. Viruses of the herpes family, which are also available from depositories such as ATCC, include, for example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV). Baculoviruses available from depositories include Autographa californica nuclear polyhedrosis virus.

[0054] As used herein, the term "transduction" refers to the ability of an AAV vector to enter one or more particular cell types and transfer the DNA contained within the AAV vector into the cell. Transduction can be assessed by measuring the amount of AAV DNA or RNA expressed from the AAV DNA in a cell or population of cells, and / or by assessing the number of cells in a population that contain AAV DNA or RNA expressed from the DNA. Where the presence or amount of RNA is assessed, the type of transduction assessed is referred to herein as "functional transduction", i.e. the ability of the AAV to transfer DNA to the cell and have that DNA expressed. Conversely,physical transduction refers simply to the transfer DNA to the cell. "Transduction efficiency" is a measure of the level of transduction from a starting amount of AAV vector (e.g. the starting amount of vector being injected in vivo or applied to cells in vitro), and can be quantitative or qualitative, and / or with reference to a particular control, e.g. a prototypic AAV vector. For example, if a candidate AAV vector transduces twice as many cells as a control vector and / or the amount of AAV DNA per cell from transduction with the candidate AAV vector is twice that of transduction with the control vector, where the starting amount of each vector was the same ( / .e. the amount of each vector injected into a subject or applied to cells was the same), it can be said that the transduction efficiency of the candidate AAV vector is 200% greater than, or is twice that of, the transduction efficiency of the control vector.

[0055] The phrase "numbering relative to" a sequence, such as SEQ ID NO: 1, means that the numbering of the amino acid position being referred to is as shown in, or is with reference to, the sequence, e.g. SEQ ID NO: 1. It will be appreciated that the sequence is simply a reference sequence, and that the same amino acid residue or position may correspond to a different numbered residue or position in a different sequence, such as if the different sequence is a truncated form or is a sequence that has insertions or deletions compared to the reference sequence or is a homolog. To identify corresponding positions or nucleotides or residues in different sequences, sequences of related or variant polynucleotides or polypeptides are aligned by any method known to those of skill in the art. Such methods typically maximize matches (e.g. identical nucleotides or amino acids at positions), and include methods such as using manual alignments and by using the numerous alignment programs available (for example, BLASTP, ClustlW, ClustlW2, EMBOSS, LALIGN, Kalign, etc.) and others known to those of skill in the art. By aligning the sequences of polypeptides, one skilled in the art can identify corresponding positions. For the purposes of the present disclosure, the recited amino acid modifications (e.g. amino acid substitutions, deletions and insertions) in the capsid polypeptides are relative to SEQ ID NO: 1.

[0056] As used herein, "corresponding nucleotides", "corresponding amino acid residues", "corresponding positions" or "positions corresponding to" and grammatical variations thereof refer to positions, nucleotides or amino acids that occur at aligned loci. The sequences of related or variant polynucleotides or polypeptides are aligned by any method known to those of skill in the art. Such methods typically maximize matches (e.g. identical nucleotides or amino acids at positions), and include methods such as using manual alignments and by using the numerous alignment programs available (for example, BLASTN, BLASTP, ClustlW, ClustlW2, EMBOSS, LALIGN, Kalign, etc) and others known to those of skill in the art. By aligning the sequences of polynucleotides or polypeptides, one skilled in the art can identify corresponding nucleotides or amino acids. For example, by aligning the prototypic AAV1 capsid polypeptide set forth in SEQ ID NO: 1 with another AAV capsid polypeptide, such as the variant set forth in SEQ ID NO: 5, as shown in Figure 2, one of skill in the art can identify regions or amino acids residues within the other AAV polypeptide that correspond to various regions or residues in the AAV polypeptide set forth in SEQ ID NO: 1. For example, the amino acid residue in SEQ ID NO: 5 that corresponds toT502 in SEQ ID NO: 1 is A501. Similarly, position 538 of SEQ ID NO: 5 corresponds to position 539 of SEQ ID NO: 1.

[0057] A "heterologous coding sequence" as used herein refers to nucleic acid sequence present in a polynucleotide, vector, or host cell that is not naturally found in the polynucleotide, vector, or host cell or is not naturally found at the position that it is at in the polynucleotide, vector, or host cell, i.e. is non-native. A "heterologous coding sequence" can encode a peptide or polypeptide, or a polynucleotide that itself has a function or activity, such as an antisense or inhibitory oligonucleotide, including antisense DNA and RNA (e.g. miRNA, siRNA, and shRNA). In some examples, the heterologous coding sequence is a stretch of nucleic acids that is essentially homologous to a stretch of nucleic acids in the genomic DNA of an animal, such that when the heterologous coding sequence is introduced into a cell of the animal, homologous recombination between the heterologous sequence and the genomic DNA can occur. In one example, the heterologous coding sequence is a functional copy of a gene for introduction into a cell that has a defective / mutated copy. In another example, the heterologous coding sequence encodes a nuclease such as a Cas9 nuclease and / or a guide RNA (gRNA), so as to effect genome editing.

[0058] As used herein, the term "operably-linked" with reference to a promoter and a coding sequence means that the transcription of the coding sequence is under the control of, or driven by, the promoter.

[0059] The term "host cell" refers to a cell, such as a mammalian cell, that has introduced into it the exogenous DNA, such as a vector or other polynucleotide. The term includes the progeny of the original cell into which the exogenous DNA has been introduced. Thus, a "host cell" as used herein generally refers to a cell that has been transfected or transduced with exogenous DNA.

[0060] As used herein, "isolated" with reference to a polynucleotide or polypeptide means that the polynucleotide or polypeptide is substantially free of cellular material or other contaminating proteins from the cells from which the polynucleotide or polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.

[0061] The term "subject" as used herein refers to an animal, in particular a mammal and more particularly a primate including a lower primate and even more particularly, a human who can benefit from the present invention. A subject, regardless of whether a human or non-human animal or embryo, may be referred to as an individual, subject, animal, patient, host or recipient. The present disclosure has both human and veterinary applications. For convenience, an "animal" specifically includes livestock animals such as cattle, horses, sheep, pigs, camelids, goats and donkeys, as well as domestic animals, such as dogs and cats. With respect to horses, these include horses used in the racing industry as well as those used recreationally or in the livestock industry. Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. In some embodiments, the subject is human.

[0062] It will be appreciated that the above-described terms and associated definitions are used for the purpose of explanation only and are not intended to be limiting.Table 1. Description of SequencesCapsid polypeptides

[0063] Provided are novel AAV capsid polypeptides, developed and identified using combinatorial capsid libraries. AAV vectors comprising the AAV capsid polypeptides display increased transduction of cells in the CNS compared to the clinically relevant AAV9 vector. Additionally, these AAV vectors can exhibit detargeting from both mouse and human hepatocytes when compared to AAV9, both in vitro and in vivo. Thus, the AAV vectors containing capsid polypeptides of the present disclosure surpass AAV9 in terms of transduction efficiency at the DNA (cell entry) and RNA (transgene expression) levels in the CNS, as well as improved CNS tropism and liver detargeting. These properties result in improved and desirable safety and CNS targeting profiles, such that the capsid polypeptides of the present disclosure have advantages and applications in gene therapy for diseases and conditions of the CNS.

[0064] The capsid polypeptides of the present disclosure are therefore particularly useful in preparing AAV vectors, such as AAV vectors for therapy of diseases or conditions of the CNS, where off-target transduction of liver cells is undesirable.

[0065] The AAV capsid polypeptides comprise a sequence set forth in in any one of SEQ ID NOs: 3-13, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. AAV capsid polypeptides of the present disclosure also include fragments of polypeptides comprising a sequence set forth in in any one of SEQ ID NOs: 3-13, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some examples, the fragment comprises at least or about 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 amino acids. In particular examples, the fragment is or comprises the VP2 region or the VP3 region of the capsid polypeptide, i.e. the VP2 region corresponding to positions 138-736 of the AAV1 capsid set forth in SEQ ID NO: 1, or the VP3 region corresponding to positions 204-736 of the AAV1 capsid set forth in SEQ ID NO: 1.

[0066] The AAV capsid polypeptides of the present disclosure comprise one or more modifications (e.g. amino acid substitutions, deletions or insertions) relative to the prototypic AAV1 capsid polypeptide set forth in SEQ ID NO: 1. The capsid polypeptides typically comprise at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 modifications compared to the prototypic AAV1 capsid polypeptide set forth in SEQ ID NO: 1. Exemplary modifications include one or more of D4G, Y6C, N14T, E21Q, D24A, D24K, A29P, A29V, K31Q, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, D41N, G42R, F56G, A67E, R92K, Q105K, V125L, L129F, G135A, G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, D178E, S179T, P185D, L188I, S205A, A263Q, T265del, S268del, T326Q, V330T, S345T, I373V, I373N, N383D, T411Q, T415E, E418N, E418D, N497Q, N498del, S499Q, N500Q, F501L, T502A, F584L, A598V, A601V, H628N, L640M, N642H, V699I, A706Y, A709T, N710S, T714A, D716N, N717T, N718E, L720V, T722S and P735N (with numbering relative to SEQ ID NO: 1), and conservative additional substitutions thereof (i.e. where the identified replacement amino acid is substituted with a different amino acid having a similar side chain as the identified replacement amino acid, e.g. when the modification is D4G, G is the identified replacement amino acid and is substituted with a different amino acid having a similar side chain to G). Put another way, the capsid polypeptides comprise one or more of G at position 4, C at position 6, T at position 14, Q at position 21, A or K at position 24, P or V at position 29, Q or K at position 31, P at position 34, A at position 35, E at position 36, R at position 37, H at position 38, K at position 39, N at position 41, R at position 42, G at position 56, E at position 67, K at position 92, K at position 105, L at position 125, F at position 129, A at position 135, A at position 141, P at position 148, insertion of R between 151 and 152, S at position 152, T at position 157, K at position 162, R at position 168, E at position 178, T at position 179, D at position 185, I at position 188, A at position 205, Q at position 263, deletion of T at position 265, deletion of S at position 268, Q at position 326, T at position 330, T at position 345, V or N at position 373, D at position 383, Q at position 411, E at position 415, N or D at position 418, Q at position 497, deletion of N at position 498, Q at position 499, Q at position 500, L at position 501, A at position 502, L at position 584, V at position 598, V at position 601, N at position 628, M at position 640, H at position 642, I at position 699, Y at position 706, T at position 709, S at position 710, A at position 714, N at position 716, T at position 717, E at position 718, V at position 720, S at position 722 and N at position 735, or conservative substitutions thereof. In a particular example, the modifications include one or more of D24A, A29V, K31Q, K38H, D41N, G42R, L129F, G135A, G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, D178E, P185D, S205A, S268del, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N497Q, N498del, S499Q, N500Q, F501L, T502A, F584L, A598V, A601V, H628N, L640M, N642H and V699I (with numbering relative to SEQ ID NO: 1), and conservative additional substitutions thereof. In a further example, the modification includes 2, 3, 4, 5, 6, 7, 8 or all of N498del, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N and T502A, or conservative additional substitutions thereof.

[0067] In particular examples, the capsid polypeptides comprise the modification N383D, N498del, and / or the modification T502A (e.g. N383D + N498del; N383D + T502A; N498del + T502A; or N383D + N498del + T502A). As can be seen from the alignment in Figure 2, there arethree sequential asparagines at positions 496, 497 and 498. Thus, as would be appreciated, any one of N496, N497 or N498 can be deleted. Accordingly, reference herein to a deletion of N498 (i.e. N498del) means deletion of any one of the three asparagines at positions 496-498 (i.e. N496del, N497del or N498del). In some examples, the capsid polypeptides comprise the modifications N498del and T502A; or N383D, N498del and T502A. Further the polypeptides may optionally include one or more of D4G, Y6C, N14T, E21Q, D24A, D24K, A29P, A29V, K31Q, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, D41N, G42S, G42R, F56G, A67E, R92K, Q105K, V125L, L129F, G135A, G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, D178E, S179T, P185D, L188I, S205A, A263Q, T265del, S268del, T326Q, V330T, S345T, I373V, I373N, N383D, T411Q, T415E, E418N, E418D, N496K, N497Q, N498del, S499Q, N500Q, F501L, T502A, F584L, A598V, A601V, H628N, L640M, N642H, V699I, A706Y, A709T, N710S, T714A, D716N, N717T, N718E, L720V, T722S and P735N; or in particular optionally one or more of T411Q, T415E and E418N; T326Q, V330T, S345T, N383D, T411Q, T415E and E418N. Thus, in some examples, the capsid polypeptides comprise the modifications N498del and T502A; N383D, N498del and T502A; T411Q, T415E, E418N, N498del and T502A; N383D, T411Q, T415E, E418N, N498del and T502A; N383D, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, and T502A; and have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the AAV capsid polypeptide set forth in any one of SEQ ID NOs: 3-12.

[0068] In a particular example, the capsid polypeptide comprises one or more of the modifications D24A, K31Q, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del or T502A. In some examples, the capsid polypeptide comprises the modifications N498del and T502A and one or more of the modifications D24A, K31Q, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E or E418N. For example, an exemplary polypeptide comprises modifications D24A, K31Q, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del and T502A, and optionally comprises the sequence set forth in SEQ ID NO: 5 (e.g. AAV- BBB6 as described herein) or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 5.

[0069] The capsid polypeptides of the present disclosure may comprise one or more of the modifications G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, P185D, S205A, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, N642H and V699I. In some examples, the capsid polypeptide comprises the modifications N498del and T502A and one or more of the modifications G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, P185D, S205A, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, 642H and V699I. For example, an exemplary polypeptide comprises modifications G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, P185D, S205A, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, N642H and V699I , and optionally comprises the sequence set forth in SEQ ID NO:8 (e.g. AAV-BBB28 as described herein) or a sequence having at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:8.

[0070] In another example, the capsid polypeptides of the present disclosure comprise one or more of the modifications D24A, G135A, Q148P, 151-152insR, E152S, S157T, T162K, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, A598V, A601V, H628N, L640M and N642H. In some examples, the capsid polypeptide comprises the modifications N498del and T502A and one or more of the modifications D24A, G135A, Q148P, 151-152insR, E152S, S157T, T162K, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, A598V, A601V, H628N, L640M and N642H. For example, an exemplary polypeptide comprises modifications D24A, G135A, Q148P, 151-152insR, E152S, S157T, T162K, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, A598V, A601V, H628N, L640M and N642H, and optionally comprises the sequence set forth in SEQ ID NO:9 (e.g. AAV-BBB31 as described herein) or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:9.

[0071] In another example, the capsid polypeptides of the present disclosure comprise one or more of the modifications D24A, Q148P, 151-152insR, E152S, S157T, T162K, D178E, S268del, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, F584L and A598V. In some examples, the capsid polypeptide comprises the modifications N498del and T502A and one or more of the modifications D24A, Q148P, 151-152insR, E152S, S157T, T162K, D178E, S268del, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, F584L and A598V. For example, an exemplary polypeptide comprises modifications D24A, Q148P, 151-152insR, E152S, S157T, T162K, D178E, S268del, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, F584L and A598V, and optionally comprises the sequence set forth in SEQ ID NO: 3 (e.g. AAV- BBB3 as described herein) or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 3.

[0072] In another example, the capsid polypeptides of the present disclosure comprise one or more of the modifications D24A, A29V, K31Q, K38H, D41N, G42R, L129F, Q148P, 151-152insR, E152S, S157T, T162K, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N496K, N497Q, N498del, S499Q, N500Q, F501L, T502A, F584L, A598V or N642H. In some examples, the capsid polypeptide comprises the modifications N498del and T502A and one or more of the modifications D24A, A29V, K31Q, K38H, D41N, G42R, L129F, Q148P, 151-152insR, E152S, S157T, T162K, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N496K, N497Q, S499Q, N500Q, F501L, F584L, A598V and N642H. For example, an exemplary polypeptide comprises modifications D24A, Q148P, 151-152insR, E152S, S157T, T162K, D178E, S268del, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N496K, N497Q, N498del, S499Q, N500Q, T502A, F584L and A598V, and optionally comprises the sequence set forth in SEQ ID NO: 13 (e.g. AAV-BBB52 as described herein) or a sequence having at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 13.

[0073] The capsid polypeptides of the present disclosure may comprise one or more of the modifications D24A, G42S, G141A, Q148P, 151-152insR, E152S, S157T, T162K, S179T, S268del, T326Q, V330T, S345T, N383D, E418D, N498del, T502A, F584L, A598V, H628N or N642H. In some examples, the capsid polypeptide comprises the modifications N498del and T502A and one or more of the modifications D24A, G42S, G141A, Q148P, 151-152insR, E152S, S157T, T162K, S179T, S268del, T326Q, V330T, S345T, N383D, E418D, F584L, A598V, H628N or N642H. For example, an exemplary polypeptide comprises modifications D24A, G42S, G141A, Q148P, 151- 152insR, E152S, S157T, T162K, S179T, S268del, T326Q, V330T, S345T, N383D, E418D, N498del, T502A, F584L, A598V, H628N and N642H, and optionally comprises the sequence set forth in SEQ ID NO:4 (e.g. AAV-BBB5 as described herein) or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:4.

[0074] In another example, the capsid polypeptides of the present disclosure comprise one or more of the modifications D4G, Y6C, D24A, A29V, K31Q, K38H, D41N, G42R, F56G, A67E, Q105K, D178E, L188I, T326Q, V330T, S345T, N383D, E418D, N498del, T502A, F584L, A598V, H628N or N642H. In some examples, the capsid polypeptide comprises the modifications N498del and T502A and one or more of the modifications D4G, Y6C, D24A, A29V, K31Q, K38H, D41N, G42R, F56G, A67E, Q105K, D178E, L188I, T326Q, V330T, S345T, N383D, E418D, F584L, A598V, H628N or N642H. For example, an exemplary polypeptide comprises modifications D4G, Y6C, D24A, A29V, K31Q, K38H, D41N, G42R, F56G, A67E, Q105K, D178E, L188I, T326Q, V330T, S345T, N383D, E418D, N498del, T502A, F584L, A598V, H628N and N642H, and optionally comprises the sequence set forth in SEQ ID NO:6 (e.g. AAV-BBB11 as described herein) or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:6.

[0075] In another example, the capsid polypeptides of the present disclosure comprise one or more of the modifications Y6C, R92K, Q105K, V125L, D178E, L188I, T326Q, V330T, S345T, N383D, E418D, N498del, T502A, F584L or A598V. In some examples, the capsid polypeptide comprises the modifications N498del and T502A and one or more of the modifications Y6C, R92K, Q105K, V125L, D178E, L188I, T326Q, V330T, S345T, N383D, E418D, F584L or A598V. For example, an exemplary polypeptide comprises modifications Y6C, R92K, Q105K, V125L, D178E, L188I, T326Q, V330T, S345T, N383D, E418D, N498del, T502A, F584L and A598V, and optionally comprises the sequence set forth in SEQ ID NO:7 (e.g. AAV-BBB27 as described herein) or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:7.

[0076] In another example, the capsid polypeptides of the present disclosure comprise one or more of the modifications D24A, A29V, K31Q, K38H, D41N, G42R, R92K, Q105K, V125L, D178E,L188I, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, F584L, A598V, N642H, V699I, A706Y, A709T, N710S, T714A, D716N, N717T, N718E, L720V, T722S or P735N. In some examples, the capsid polypeptide comprises the modifications N498del and T502A and one or more of the modifications D24A, A29V, K31Q, K38H, D41N, G42R, R92K, Q105K, V125L, D178E, L188I, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, F584L, A598V, N642H, V699I, A706Y, A709T, N710S, T714A, D716N, N717T, N718E, L720V, T722S or P735N. For example, an exemplary polypeptide comprises modifications D24A, A29V, K31Q, K38H, D41N, G42R, R92K, Q105K, V125L, D178E, L188I, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, F584L, A598V, N642H, V699I, A706Y, A709T, N710S, T714A, D716N, N717T, N718E, L720V, T722S and P735N, and optionally comprises the sequence set forth in SEQ ID NO: 11 (e.g. AAV-BBB38 as described herein) or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 11.

[0077] In another example, the capsid polypeptides of the present disclosure comprise one or more of the modifications Q105K, V125L, T162K, D178E, T326Q, V330T, S345T, I373N, N383D, T411Q, T415E, E418N, N498del, T502A, F584L or A598V. In some examples, the capsid polypeptide comprises the modifications N498del and T502A and one or more of the modifications Q105K, V125L, T162K, D178E, T326Q, V330T, S345T, I373N, N383D, T411Q, T415E, E418N, F584L and A598V. For example, an exemplary polypeptide comprises modifications Q105K, V125L, T162K, D178E, T326Q, V330T, S345T, I373N, N383D, T411Q, T415E, E418N, N498del, T502A, F584L and A598V, and optionally comprises the sequence set forth in SEQ ID NO: 12 (e.g. AAV-BBB50 as described herein) or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 12.

[0078] In another example, the capsid polypeptides of the present disclosure comprise one or more of the modifications N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G141A, Q148P, 151-152insR, E152S, S157T, T162K, S179T, A263Q, T265del, T326Q, V330T, S345T, I373V, E418D, N642H, V699I, A706Y, A709T, N710S, T714A, D716N, N717T, N718E, L720V, T722S or P735N. In some examples, the capsid polypeptide comprises all modifications N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G141A, Q148P, 151-152insR, E152S, S157T, T162K, S179T, A263Q, T265del, T326Q, V330T, S345T, I373V, E418D, N642H, V699I, A706Y, A709T, N710S, T714A, D716N, N717T, N718E, L720V, T722S and P735N, and optionally comprises the sequence set forth in SEQ ID NO: 10 (e.g. AAV- BBB37 as described herein) or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 10.

[0079] The capsid polypeptides may also comprise one or more modifications that confer increased immune evasion on an AAV vector comprising a capsid comprising the polypeptide. Such modifications may, for example, disrupt epitopes in the capsid that are recognised by antibodies or T cells (e.g. cytotoxic T cells). Non-limiting examples of modifications for enhancingimmune evasion include replacement of 456-AQNK-459 with 456-SERR-459, and replacement of 588-STDPATGDVH-597 with 588-DLDPKATEVE-597, with numbering relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

[0080] The transduction of cells of the CNS by AAV vectors having a capsid comprising a capsid polypeptide of the present disclosure is generally increased or enhanced compared to AAV vectors comprising a reference AAV capsid polypeptide (e.g. the AAV1 or AAV9 capsid of SEQ ID NO: 1 or 2, respectively). Transduction or transduction efficiency of the AAV vectors can be increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. For example, an AAV vector comprising a capsid polypeptide of the present disclosure can be at least or about 1.2x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, llx, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx or more efficient at transducing cells in vivo compared to an AAV vector comprising a reference AAV capsid polypeptide e.g. the AAV9 capsid of SEQ ID NO:2). The cell of the CNS may be in the brainstem, cerebellum, midbrain, thalamus, spinal cord, cortex or hippocampus. In particular examples, the cells is in the brainstem, cerebellum, midbrain, thalamus or spinal cord. The cell may be a neuron (e.g. a Purkinje cell, basket cell, Betz cell, Lugaro cell, medium spiny neuron, pyramidal cell, rosehip cell, Renshaw cell, unipolar brush cell, granule cell, anterior horn cell or spindle cell) or glial cell (e.g. an astrocyte, oligodendrocyte, ependymal cell, or microglia).

[0081] Importantly, the transduction of cells outside of the CNS (in particular the liver but also heart, kidney or muscle) by AAV vectors having a capsid comprising a capsid polypeptide of the present disclosure is generally decreased compared to AAV vectors comprising a reference AAV capsid polypeptide (e.g. the AAV1 or AAV9 capsid of SEQ ID NO: 1 or 2, respectively). Transduction or transduction efficiency of the AAV vectors of cells outside of the CNS (e.g. cells in the liver, heart, kidney or muscle) can be decreased by at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to AAV vectors comprising a reference AAV capsid polypeptide (e.g. the AAV1 or AAV9 capsid of SEQ ID NO: 1 or 2, respectively). In some examples, the cells are hepatocytes.

[0082] Also provided are nucleic acid molecules, including isolated nucleic acid molecules, encoding a capsid polypeptide of the disclosure. Thus, amongst the nucleic acid molecules provided herein are those encoding a capsid polypeptide comprising the VP1, VP2 and / or VP3 of any one of the capsid polypeptides set forth in SEQ ID NOs: 3-13 as described above or a polypeptides having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Nonlimiting examples of nucleic acid molecules therefore include those set forth in SEQ ID NOs: 14- 24, those having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and those that hybridize with medium or high stringency to nucleic acid molecules comprising a sequence set forth in any one of SEQ ID NOs: 14-24.Vectors

[0083] The present disclosure also provides vectors comprising a nucleic acid molecule that encodes a capsid polypeptide described herein, and vectors comprising a capsid polypeptide described herein. The vectors include nucleic acid vectors that comprise a nucleic acid molecule that encodes a capsid polypeptide described herein, and AAV vectors that have a capsid comprising a capsid polypeptide described herein.Nucleic acid vectors

[0084] Vectors of the present disclosure include nucleic acid vectors that comprise a polynucleotide that encodes all or a portion of a capsid polypeptide described herein. In some examples, the vectors comprise a sequence set forth in any one of SEQ ID Nos: 14-24. The vectors can be episomal vectors ( / .e., that do not integrate into the genome of a host cell) or can be vectors that integrate into the host cell genome. Exemplary vectors that comprise a nucleic acid molecule encoding a capsid polypeptide include, but are not limited to, plasmids, cosmids, transposons and artificial chromosomes. In particular examples, the vectors are plasmids.

[0085] Vectors, such as plasmids, suitable for use in bacterial, insect and mammalian cells are widely described and well-known in the art. Those skilled in the art would appreciate that vectors of the present disclosure may also contain additional sequences and elements useful for the replication of the vector in prokaryotic and / or eukaryotic cells, selection of the vector and the expression of a heterologous sequence in a variety of host cells. For example, the vectors of the present disclosure can include a prokaryotic replicon (that is, a sequence having the ability to direct autonomous replication and maintenance of the vector extra-chromosomally in a prokaryotic host cell, such as a bacterial host cell). Such replicons are well known in the art. In some embodiments, the vectors can include a shuttle element that makes the vectors suitable for replication and integration in both prokaryotes and eukaryotes. In addition, vectors may also include a gene whose expression confers a detectable marker such as a drug resistance gene, which allows for selection and maintenance of the host cells. Vectors may also have a reportable marker, such as gene encoding a fluorescent or other detectable protein. The nucleic acid vectors will likely also comprise other elements, including any one or more of those described below. Most typically, the vectors will comprise a promoter operably linked to the nucleic acid encoding the capsid protein.

[0086] The nucleic acid vectors of the present disclosure can be constructed using known techniques, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, in vitro or chemical synthesis of DNA, and DNA sequencing. The vectors of the present disclosure may be introduced into a host cell using any method known in the art. Accordingly, the present disclosure is also directed to host cells comprising a vector or nucleic acid described herein.AA V vectors

[0087] Provided herein are AAV vectors comprising a capsid polypeptide described herein. Methods for vectorizing a capsid protein are well known in the art and any suitable method can be employed for the purposes of the present disclosure. For example, the cap gene can be recovered (e.g. by PCR or digest with enzymes that cut upstream and downstream of cap) and cloned into a packaging construct containing rep. Any AAV rep gene may be used, including, for example, a rep gene is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13 and any variants thereof. Typically, the cap gene is cloned downstream of rep so the rep p40 promoter can drive cap expression. This construct does not contain ITRs. This construct is then introduced into a packaging cell line with a second construct containing ITRs, typically flanking a heterologous coding sequence. Helper function or a helper virus are also introduced, and recombinant AAV comprising a capsid generated from capsid proteins expressed from the cap gene, and encapsidating a genome comprising the transgene flanked by the ITRs, is recovered from the supernatant of the packaging cell line. Various types of cells can be used as the packaging cell line. For example, packaging cell lines that can be used include, but are not limited to, HEK293 cells, HeLa cells, and Vero cells, for example as disclosed in US20110201088. The helper functions may be provided by one or more helper plasmids or helper viruses comprising adenoviral helper genes. Non-limiting examples of the adenoviral helper genes include E1A, E1B, E2A, E4 and VA, which can provide helper functions to AAV packaging. Helper viruses of AAV are known in the art and include, for example, viruses from the family Adenoviridae and the family Herpesviridae. Examples of helper viruses of AAV include, but are not limited to, SAdV-13 helper virus and SAdV-13-like helper virus described in US20110201088, helper vectors pHELP (Applied Viromics). A skilled artisan will appreciate that any helper virus or helper plasmid of AAV that can provide adequate helper function to AAV can be used herein.

[0088] In some instances, rAAV virions are produced using a cell line that stably expresses some of the necessary components for AAV virion production. For example, a plasmid (or multiple plasmids) comprising the nucleic acid containing a cap gene identified as described herein and a rep gene, and a selectable marker, such as a neomycin resistance gene, can be integrated into the genome of a cell (the packaging cells). The packaging cell line can then be transfected with an AAV vector and a helper plasmid or transfected with an AAV vector and co-infected with a helper virus (e.g., adenovirus providing the helper functions). The advantages of this method are that the cells are selectable and are suitable for large-scale production of the recombinant AAV. As another non-limiting example, adenovirus or baculovirus rather than plasmids can be used to introduce the nucleic acid encoding the capsid polypeptide, and optionally the rep gene, into packaging cells. As yet another non-limiting example, the AAV vector is also stably integrated into the DNA of producer cells, and the helper functions can be provided by a wild-type adenovirus to produce the recombinant AAV.

[0089] In still further instances, the AAV vectors are produced synthetically, by synthesising AAV capsid proteins and assembling and packaging the capsids in vitro.

[0090] Typically, the AAV vectors of the present disclosure also comprise a heterologous coding sequence. The heterologous coding sequence may be operably linked to a promoter tofacilitate expression of the sequence. The heterologous coding sequence can encode a peptide or polypeptide, such as a therapeutic peptide or polypeptide, or can encode a polynucleotide or transcript that itself has a function or activity, such as an antisense or inhibitory oligonucleotide, including antisense DNA and RNA (e.g. miRNA, siRNA, shRNA or gRNA). In some examples, the heterologous coding sequence is a stretch of nucleic acids that is essentially homologous to a stretch of nucleic acids in the genomic DNA of an animal, such that when the heterologous coding sequence is introduced into a cell of the animal, homologous recombination between the heterologous coding sequence and the genomic DNA can occur. As would be appreciated, the nature of the heterologous coding sequence is not essential to the present disclosure. In particular embodiments, the vectors comprising the heterologous coding sequence(s) will be used in gene therapy.

[0091] In particular examples, the heterologous coding sequence encodes a peptide or polypeptide, or polynucleotide, whose expression is of therapeutic use, such as, for example, for the treatment of a disease or disorder. For example, expression of a therapeutic peptide or polypeptide may serve to restore or replace the function of the endogenous form of the peptide or polypeptide that is defective ( / .e. gene replacement therapy). In other examples, expression of a therapeutic peptide or polypeptide, or polynucleotide, from the heterologous sequence serves to alter the levels and / or activity of one or more other peptides, polypeptides or polynucleotides in the host cell. Thus, according to particular embodiments, the expression of a heterologous coding sequence introduced by a vector described herein into a host cell can be used to provide a therapeutic amount of a peptide, polypeptide or polynucleotide to ameliorate the symptoms of a disease or disorder. In other instance, the heterologous coding sequence is a stretch of nucleic acids that is essentially homologous to a stretch of nucleic acids in the genomic DNA of an animal, such that when the heterologous sequence is introduced into a cell of the animal, homologous recombination between the heterologous coding sequence and the genomic DNA can occur. Accordingly, the introduction of a heterologous sequence by an AAV vector described herein into a host cell can be used to correct mutations in genomic DNA, which in turn can ameliorate the symptoms of a disease or disorder.

[0092] In non-limiting examples, the heterologous coding sequence encodes an expression product that, when delivered to a subject using an AAV vector of the present disclosure, treats a disease or condition of the CNS ( / .e. a disease or condition with a pathology that manifests at least in part in the CNS, and / or is caused at least in part by expression of one or more genes in the CNS). For example, the disease or condition may be a demyelinating disease or a tumour. In illustrative embodiments, the disease or condition is selected from genetic epilepsies (e.g. Dravet syndrome, Lennox-Gastaut syndrome, West syndrome, and Angelman syndrome), AADC Deficiency, Parkinson's disease, Canavan's disease, Niemann-Pick disease, Alzheimer's disease, Huntington's disease, Batten Disease, Giant axonal neuropathy, Adrenoleukodystrophy, Rett Syndrome, Spinal muscular atrophy, Multiple system atrophy, Amyotrophic lateral sclerosis, Krabbe disease, Tay-Sachs disease, Mucopolysaccharidosis (e.g. MPS I, MPS II, MPS IIIA, MPS IIIB, MPS VI), Ceroid lipofuscinoses (e.g. CLN1, CLN2, CLN3, CLN5, CLN6, CLN7, CLN8, CLN10,CLN11), Metachromatic leukodystrophy, and gangliosidosis. Those skilled in the art would readily be able to select an appropriate heterologous coding sequence useful for treating such central nervous system-associated diseases and conditions. In some examples, the heterologous coding sequence comprises all or a part of a gene that is associated with the disease. In such instances, the AAV vector is typically used for gene replacement therapy in which a functional copy of the gene is introduced, or for genome editing (e.g. CRISPR-Cas9 based genome editing) in which one or more defective copies of a gene is corrected or ablated. In other instances, the heterologous coding sequence encodes a therapeutic protein that is not associated with the disease (i.e. is not causative of the disease in the subject).

[0093] The heterologous coding sequence in the AAV vector is flanked by 3' and 5' AAV ITRs. AAV ITRs used in the vectors of the disclosure need not have a wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13. Such ITRs are well known in the art.

[0094] As will be appreciated by a skilled artisan, any method suitable for purifying AAV can be used in the embodiments described herein to purify the AAV vectors, and such methods are well known in the art. For example, the AAV vectors can be isolated and purified from packaging cells and / or the supernatant of the packaging cells. In some embodiments, the AAV is purified by separation method using a CsCI or iodixanol gradient centrifugation. In other embodiments, AAV is purified as described in US20020136710 using a solid support that includes a matrix to which an artificial receptor or receptor-like molecule that mediates AAV attachment is immobilized.Additional elements in the vectors

[0095] The vectors of the present disclosure can comprise promoters. In instances where the vector is a nucleic acid vector comprising nucleic acid encoding the capsid polypeptide, the promoter may facilitate expression of the nucleic acid encoding the capsid polypeptide. In instances where the vector is an AAV vector, the promoter may facilitate expression of a heterologous coding sequence, as described above.

[0096] In some examples, the promoters are AAV promoters, such as the p5, pl9 or p40 promoter. In other examples, the promoters are derived from other sources. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the [3-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter. Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicatingcells only. Non-limiting examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system; the ecdysone insect promoter, the tetracycline-repressible system, the tetracyclineinducible system, the RU486-inducible system and the rapamycin-inducible system. Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only. In some embodiments, tissue specific promoters are used. In some embodiments, the promoters selectively function in the brain, and may include, for example, human synapsin 1 (Synl) promoter, neuron-specific enolase (NSE) promoter, human myelin associated (MAG) promoter (see e.g. Ingusci et al., 2019, Front Pharmacol 10, 724. doi: 10.3389 / fphar.2019.00724). The selection of an appropriate promoter is well within the ability of one of ordinary skill in the art.

[0097] The vectors can also include transcriptional enhancers, translational signals, and transcriptional and translational termination signals. Examples of transcriptional termination signals include, but are not limited to, polyadenylation signal sequences, such as bovine growth hormone (BGH) poly(A), SV40 late poly(A), rabbit beta-globin (RBG) poly(A), thymidine kinase (TK) poly(A) sequences, and any variants thereof. In some embodiments, the transcriptional termination region is located downstream of the posttranscriptional regulatory element. In some embodiments, the transcriptional termination region is a polyadenylation signal sequence.

[0098] The vectors can include various posttranscriptional regulatory elements. In some embodiments, the posttranscriptional regulatory element can be a viral posttranscriptional regulatory element. Non-limiting examples of viral posttranscriptional regulatory element include woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), hepatitis B virus posttranscriptional regulatory element (HBVPRE), RNA transport element, and any variants thereof. The RTE can be a rev response element (RRE), for example, a lentiviral RRE. A nonlimiting example is bovine immunodeficiency virus rev response element (RRE). In some embodiments, the RTE is a constitutive transport element (CTE). Examples of CTE include, but are not limited to, Mason-Pfizer Monkey Virus CTE and Avian Leukemia Virus CTE.

[0099] A signal peptide sequence can also be included in the vector to provide for secretion of a polypeptide from a mammalian cell. Examples of signal peptides include, but are not limited to, the endogenous signal peptide for HGH and variants thereof; the endogenous signal peptide for interferons and variants thereof, including the signal peptide of type I, II and III interferons and variants thereof; and the endogenous signal peptides for known cytokines and variants thereof, such as the signal peptide of erythropoietin (EPO), insulin, TGF-pi, TNF, ILl-a, and IL1- 8, and variants thereof. Typically, the nucleotide sequence of the signal peptide is located immediately upstream of the heterologous sequence (e.g., fused at the 5' of the coding region of the protein of interest) in the vector.

[0100] In further examples, the vectors can contain a regulatory sequence that allows, for example, the translation of multiple proteins from a single mRNA. Non-limiting examples of such regulatory sequences include internal ribosome entry site (IRES) and 2A self-processing sequence, such as a 2A peptide site from foot-and-mouth disease virus (F2A sequence).Host cells

[0101] Also provided herein are host cells comprising a nucleic acid molecule or vector (including an AAV vector) of the present disclosure. In some instances, the host cells are used to select, amplify, replicate, package and / or purify a polynucleotide or vector. In other examples, the host cells are used to express a heterologous sequence, such as one packaged within an AAV vector. Exemplary host cells include prokaryotic and eukaryotic cells. In some instances, the host cell is a mammalian host cell, such as a human host cell. It is well within the skill of a skilled artisan to select an appropriate host cell for the expression, amplification, replication, packaging and / or purification of a polynucleotide, vector or rAAV virion of the present disclosure. Exemplary mammalian host cells include cells of the CNS, in particular neurons and glial cells (e.g. an astrocyte, oligodendrocyte, ependymal cell, or microglia).Compositions and methods

[0102] Also provided are compositions comprising the nucleic acid molecules, polypeptides and / or vectors of the present disclosure. In particular examples, provided are pharmaceutical compositions comprising the AAV vectors disclosed herein and a pharmaceutically acceptable carrier. The compositions can also comprise additional ingredients such as diluents, stabilizers, excipients, and adjuvants.

[0103] The carriers, diluents and adjuvants can include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides (e.g., less than about 10 residues); proteins such as serum aAAVC.umin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween™, Pluronics™ or polyethylene glycol (PEG). In some embodiments, the physiologically acceptable carrier is an aqueous pH buffered solution.

[0104] The AAV vectors of the present disclosure, and compositions containing the AAV vectors, may be used in methods for the introduction of a heterologous coding sequence into a host cell. Such methods involve contacting the host cell with the AAV vector. This may be performed in vitro, ex vivo or in vivo. In particular embodiments, the host cell is a cell of the CNS, in particular a neuron or glial cell (e.g. an astrocyte, oligodendrocyte, ependymal cell, or microglia).

[0105] When the methods are performed ex vivo or in vivo, typically the introduction of the heterologous sequence into the host cell is for therapeutic purposes, whereby expression of the heterologous sequence results in the treatment of a disease or condition. Thus, the AAV vectors disclosed herein can be administered to a subject e.g., a human) in need thereof, such as subject with a disease or condition amendable to treatment with a protein, peptide or polynucleotide encoded by a heterologous sequence described herein.

[0106] When used in vivo, titres of AAV vectors to be administered to a subject will vary depending on, for example, the particular recombinant virus, the disease or disorder to be treated, the mode of administration, the treatment goal, the individual to be treated, and the cell type(s) being targeted, and can be determined by methods well known to those skilled in the art. Although the exact dosage will be determined on an individual basis, in most cases, typically, recombinant viruses of the present disclosure can be administered to a subject at a dose of between lxlO10genome copies of the recombinant virus per kg of the subject and lxlO14genome copies per kg. In other examples, less than lxlO10genome copies may be sufficient for a therapeutic effect. In other examples, more than lxlO14genome copies may be required for a therapeutic effect.

[0107] The route of the administration is not particularly limited. For example, a therapeutically effective amount of the AAV vector can be administered to the subject via, for example, intravenous, intrathecal, intracerebral, intraparenchymal, intracerebroventricular, intranasal, intraperitoneal, intravitreal, subcutaneous, epicutaneous, intradermal, intramuscular, pulmonary, intraosseous, oral, or buccal routes. In particular embodiments, the AAV vector is administered to the subject via intravenous, intrathecal, intracerebral, intraparenchymal, intracerebroventricular or intranasal administration. The AAV vector can be administrated as a single dose or multiple doses, and at varying intervals.

[0108] Also provided are methods for producing an AAV vector described above and herein, i.e. one comprising a capsid polypeptide of the present disclosure. Such methods comprise culturing a host cell comprising a nucleic acid molecule encoding a capsid polypeptide the present disclosure, an AAV rep gene, a heterologous coding sequence flanked by AAV inverted terminal repeats, and helper functions for generating a productive AAV infection, under conditions suitable to facilitate assembly of an AAV vector comprising a capsid comprising a capsid polypeptide of the present disclosure, wherein the capsid encapsidates the heterologous coding sequence.

[0109] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

[0110] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.ExamplesExample 1. Materials and MethodsCell lines

[0111] Adherent human embryonic kidney (HEK) 293T cell line (ATCC, Cat#CRL-3216) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Cat#11965) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich Cat#F9423), 1 x penicillinstreptomycin (PS) (Gibco, Cat#15070), and grown in a humidified incubator at 37°C with 5% CO2.

[0112] The Chinese Hamster Ovary (CHO) cell line variants Pro5 and Lec2 were a generous gift from Dr Grant Logan, Gene Therapy Research Unit, CMRI, and were grown as adherent cultures in minimum essential medium a (MEM-a) (Gibco, Cat#12571) supplemented with 10% FBS, and 1 x PS. Cells were maintained in a humidified incubator at 37°C with 5% CO2.Animals

[0113] All animal care and experimental procedures were approved by the joint Animal Care and Ethics Committee of Children's Medical Research Institute (CMRI) and The Children's Hospital at Westmead.

[0114] Adult male BALB / cJ mice (6 - 8 weeks of age) were purchased from Australian BioResources (ABR). Mice underwent a 1-week acclimatisation period to relocation to CMRI Animal Facility and were subsequently housed in standard conditions in a 12-h light : 12-h dark environment. CMRI's established colony of Fah_ / “ / Rag2“ / “ / II2rg_ / “ (FRG) mice was used to breed naive recipient FRG animals. FRG mice were housed in individually ventilated cages with 2-(2- nitro-4-trifluoro-methylbenzoyl)-l,3-cyclohexanedione (NTBC) supplemented in drinking water (8 mg / mL). Female FRG mice (6 - 8 weeks of age) were engrafted with primary human hepatocytes (Lonza, Basel, Switzerland) as described previously (Azuma et al. Nat Biotechnol. 2007;25(8):903-910). Humanized FRG (hFRG) mice were placed on 10% NTBC prior to transduction with vectors and were maintained on 10% NTBC until harvest.Animal injections

[0115] The viral library was injected into naive male FRG mice through intravenous route (lateral tail vein) (N = 1) at 5 x 1011vg in round 1 and 2 of selection, and at 2 x 1011vg in round 3 and 4. For each selection round, three weeks post injection, mice were euthanized following by perfusion with cold HBSS (Thermo Fisher, Cat #14025092). Whole brain was then harvested and snap frozen in liquid nitrogen before being stored at -80°C.

[0116] For NGS screening of the 'AAV-BBB Testing Kit', naive male FRG (6-8 weeks old) were randomly selected and injected intravenously (lateral tail vein) with the indicated vectors at a dose of 1.5 x 1010vg per vector (1.8 x 1011total vg). Four weeks post injection, mice wereeuthanized and transcardially perfused with HBSS. The brain and spinal cord were harvested for downstream analysis.

[0117] To compare vector transduction of individual AAV variants in different strains of mice, 5 x 1011vg of each AAV was individually injected intravenously (lateral tail vein) into hFRG mice, naive male FRG (6-8 weeks old) and male BALB / cJ mice (6-8 weeks old). Three weeks post injection, mice were euthanized and transcardially perfused with HBSS. For male FRG mice, brain, spinal cord, liver, kidney, heart and muscle were harvested. For BALB / cJ mice, brain, spinal cord and liver were harvested for downstream analysis.Shuffle AAV capsid library generation and library selection

[0118] The AAV library was generated as previously described using AAV variants 1 - 12 in the parental mix (referred to as pRC-AAVLib_l-12) (Cabanes-Creus et al. Mol Ther Methods Clin Dev. 2018;12:71-84). To move this library from a replication competent backbone into a functional transduction (FT) platform (Westhaus et al. Hum Gene Ther. 2022;33(ll-12):664- 682), the pRC-AAVLib_ 1-12 was digested overnight alongside the pFT-SFFV backbone with Swal (NEB, Cat # R0604S) and Nsil (NEB, Cat # R0127S). Following separation on 1% (w / v) agarose gel and purification using Zymoclean Gel DNA Recovery Kit (Zymogen, Cat# D4001), 1.4 pg of the insert library was ligated with lpg of the linearised pFT-SFFV-eGFP platform backbone at 16°C for 16 h using T4 DNA ligase (NEB, Cat# M0202). Ligation reactions were concentrated by ethanol precipitation and electroporated into SS320 electro-competent bacteria (Lucigen, Cat# 60512). The recovered transformants were used to inoculate 250 mL of lysogeny broth (LB) containing 10 pg / mL trimethoprim (TMP). Total FT-SFFV-eGFP-AAVLib_l - 12 library plasmid was purified using the EndoFree Maxiprep Kit (Invitrogen, Cat# A31217).

[0119] For genomic DNA (gDNA) extraction from mouse brain tissue, standard phenol:chloroform protocol was used after proteinase K and RNase A digestion, as previously described without modification (Westhaus et al. Hum Gene Ther. 2020;31(9-10):575-589). The cap sequences from the extracted DNA were amplified by PCR using the Cap_Recovery_F / R primers (Table S2) and cloned directly using Gibson assembly into recipient plasmid as previously described (Cabanes-Creus et al. Mol Ther Methods Clin Dev. 2021;24:88-101). AAV capsid ORFs (cap) from round 4 of library selection were cloned into standard packaging plasmid downstream of AAV2 rep using Gibson assembly. N = 50 randomly selected clones were sent for Sanger sequencing of the capsid coding region at the Garvan Molecular Genetics facility of the Garvan Institute of Medical Research (Darlinghurst, NSW, Australia) using primers Externa l_Seq_F / R and internal_Cap_Seq (Table S2). To allow for visualization of parental contribution in selected capsids, the Xover online tool was used to create cross-over maps (Huang et al. Biotechniques. 2016;60(2):91-94).AAV viral production and titration

[0120] All AAV vectors were produced in adherent HEK293T cells by triple-transfection using PEI MAX (Polysciences, Cat# 24765-1) as previously described (Xiao X et al. J Virol.1998;72(3):2224-2232). Briefly, triple co-transfection of the pRep2 / Cap plasmid (7.5 pg per dish), pAd5 Helper plasmid (22 pg per dish), and the transgene plasmid (7.5 pg per dish) was performed in 15 cm dishes seeded with HEK293T cells. A modified transfection protocol was used for the AAV capsid libraries. 200 ng of FT-SFFV-AAVLib_l-12 library plasmid was used per dish to minimise cross-packaging (under 10%) as per our previously published protocol (Westhaus et al. Hum Gene Ther. 2022;33(ll-12):664-682). In replacement of the transgene plasmid, pRep2 helper plasmids (5 pg per dish) was transfected alongside pAd5 Helper plasmid (22 pg per dish). Three days post-transfection, recombinant virus was harvested from the cells and media, and purified using iodixanol gradient ultracentrifugation as previously described (Khan et al. Nat Protoc. 2011;6(4):482-501) and concentrated using Amicon Ultra-4 Centrifuge Filter Units with Ultracel-100 kDa membrane (EMD Millipore, Cat# UFC810024). AAV preparations were quantified by droplet digital PCR (ddPCR) (Bio-Rad, Berkeley, CA, USA) using EvaGreen supermix (Bio-Rad, Cat# 1864034) and following the manufacturer's instructions using mCherry or GFP-specific primers.Cloning of AAV transgene cassettes

[0121] The three single-stranded (ss) rAAV genomes used in this study were: (i) pCMV- mCherry-WPRE-BGHpA, containing the fluorescent protein mCherry under control of the ubiquitous CMV promoter; (ii) pCMV-eGFP-WPRE-BGHpA, containing the fluorescent protein, eGFP, under control of the CMV promoter; and (iii) phSyn-mCherry-NeBC-WPRE, containing the fluorescent protein, mCherry, under control of the human synapsin promoter and harbouring a unique 6-mer barcode sequence located between mCherry and WPRE to differentiate unique capsids packaging the same transgene. For the LY6A experiments, Ly6a expression vector was cloned into the pAAV-CMV-eGFP-WPRE-BGHpA plasmid. EcoRI-HF (NEB, Cat# R3101S) and Hindlll-HF (NEB, Cat# R3104S) restriction enzymes were used for the plasmid backbone and the Ly6a cDNA synthesized as gBIocks (IDT). Both fragments were isolated, ligated with T4 ligase (NEB) and transformed in DH5a competent cells. Construct was confirmed by Sanger sequencing.PCR BC amplification and Next Generation Sequencing (NGS)

[0122] Isolation of DNA and RNA, and cDNA synthesis was performed as described previously (Cabanes-Creus et al. Mol Ther Methods Clin Dev. 2021;24:88-101). Briefly, total DNA was extracted using standard phenol:chloroform methods, and RNA was extracted using the Direct-Zol kit (Zymogen Cat# R2062). cDNA synthesis was performed using the Superscript IV First-Strand Synthesis System (Invitrogen, Cat# 18091050) according to manufacturer's instructions with Oligo(dT)'s used for priming. 500 ng of RNA was used as input for the cDNA reaction.

[0123] For amplification and recovery of the AAV BC region for NGS analysis, 100 ng of extracted total DNA or 3 pL cDNA product was amplified using one of four available forward primers (BC_F_l-4; barcoded to allow multiplexing of different samples) and a universal reverse primer (BC_R). PCR was performed using the Q5 high-fidelity DNA polymerase (NEB, Cat#M0491L). To analyse capsid enrichment, NGS reads from the DNA and cDNA populations were normalized to the reads from the pre-injection mix and displayed as a percentage of total reads.Immunohistochemistry

[0124] To prepare tissue for analysis of native mCherry fluorescence, tissue samples were fixed with 4% (w / v in PBS) paraformaldehyde (PFA) overnight at 4°C before being cryoprotected through a sucrose gradient (10%, 20% and 30% w / v sucrose in PBS). Tissue samples were then frozen in optimal cutting temperature O.C.T. (Tissue-Tek; Sakura Finetek USA, Torrance, CA) and sectioned using a Cryostat (Leica, Cat# CM1950). O.C.T embedded tissues were sectioned at a thickness of 50 pm for brain, 15 pm for spinal cord, 5 pm for the liver and 20 pm for all other tissues. Tissues were stained with DAPI (Invitrogen, D1306) at 1: 1,000 for 5 min prior to imaging.

[0125] For immunostaining of brain, sectioned tissues were first permeabilized for 10 min with with 0.2% Triton X-100 in PBS (PBS-T) before blocking at room temperature for 1 h in blocking buffer (10% normal donkey serum in 0.2% PBS-T). Incubation in primary antibody rabbit anti-NeuN (1:200, Abeam, abl77487), rabbit anti-GFAP (1:200, DAKO, Z0334) was then performed for 20-24 h at room temperature or overnight at 4°C for primary rabbit anti-olig2 antibody (1:200, Abeam, abl09186) in blocking buffer. Sections were washed 3x with PBS with 0.1% Tween before incubation in goat anti-rabbit Alexa Fluor Plus 488 secondary antibody (1:500, Thermo Fisher, A32731) or donkey anti-rabbit Alexa Fluor Plus 647 secondary antibody (1:500, Thermo Fisher, A32795) diluted in blocking buffer for 2 h at room temperature. For immunostaining of spinal cord sections, the same protocol was used, however, only staining with NeuN was performed.

[0126] For immunostaining of chimeric liver samples, one lobe was collected prior liver perfusion, and liver sections (5 pm) were prepared on a Cryostat (Leica, Cat# CM1950). Sections were permeabilized with 0.1% PBS-T and blocked in 10% rabbit serum (Sigma Aldrich), 9% FBS in PBS for 1 h at room temperature. Sections were then incubated with rabbit monoclonal antihuman GAPDH antibody conjugated with Alexa Fluor 647 (1:650, Abeam, ab215227, clone AF674) at room temperature for 2 h.Imaging and quantification

[0127] Images were captured and analyzed on a Zeiss Axio Imager.Ml using ZEN 2 software. For quantitative image analysis, images were captured on a Leica Stellaris 8 confocal microscope using a 20x objective. mCherry positive NeuN cells analysis was performed with Cel IProfiler v4.2.5. All acquired images were then processed on Fiji.Biodistribution analysis

[0128] Vector genome copy numbers (VCN) from mouse tissues were determined by ddPCR after extraction of total DNA using DNeasy Blood and Tissue kit (QIAGEN, Cat# 69504).Vector genome content in each tissue was determined by digesting 60 ng of DNA per reaction with EcoRI-HF (NEB, Cat# R3101S) and quantified by ddPCR using a primer set and probe designed specifically for mCherry or GFP and mouse acting (mActing) as a reference (primer and probe sequences set forth in Table 2.ELISA measurement of anti -AAV IgG-specific antibody titer in human serum

[0129] Eight human sera were assayed for reactivity to AAV9, AAV-BBB6, AAV-BBB28 and AAV-BBB31 by ELISA as described in detail previously with no modifications (Cabanes-Creus et al. Mol Ther Methods Clin Dev. 2023;28:220-237).Model building and structure visualisation

[0130] The models of AAV-BBB6, AAV-BBB28, and AAV-BBB31 variant VP3 monomers were built by uploading their individual sequences to the protein modelling server SWISS-MODEL with the structure of AAV1 [Protein Data Bank (PDB) ID: 6JCR] as the template. Monomers visualization and superposition were generated using the software USCF ChimeraX.IVIg neutralization assay

[0131] Capsid reactivity with human IVIg was assessed in HEK293T cells. First, 1.5 x 105HEK293T cells per well (24-well format) were seeded 24 h before transduction with capsids to be assessed (multiplicity of transduction (MOT) of 50,000 vg per cell). For neutralisation assay, vectors were diluted in DMEM solution and incubated for lh at 37°C with increasing doses of human IVIg serum (undiluted, 1: 1, 1:2, 1:4, 1:8, 1: 16. 1:32 and no IVIg control) (Intragam® 10, 10 g / lOOmL, CSL Behring). The individual dose mixtures of vector and IVIg were then added to the cells, with a full media change of complete DMEM performed 16 h post transduction. Cells were incubated for a further 32 h (48 h total) before harvesting. Transduction efficiency was assayed by flow cytometry (BD FACSCanto cell analyzer, BD Bioscience) by determining the percentage of GFP-positive cells. All readouts were normalized to controls with no IVIg treatment.CHO cell surface binding and Transduction Assays

[0132] For surface binding assays, Chinese hamster ovary (CHO) cells were seeded in standard TC-coated 24-well plates (3 x 105 / well). The following day the plates were pre-incubated in complete medium at 4°C for 1 h. Respective AAV variants were applied at a MOT of 10,000 vg per cell. The plate was subsequently incubated at 4°C for 1 h. Cells were washed three times with cold PBS to ensure removal of unbound AAV particles. Cells were then lysed and harvested for total DNA extraction (including AAV viral genome DNA in cell surface-bound viral particles) for ddPCR analyses. Vector genomes bound were normalized to the number of cell genomes by using primers specific to CHO-gactin (Table S2).

[0133] For transduction assays, AAV variants were added to CHO cells 24-well plates (1.5 x 105 / well) at a MOT of 10,000 vg per cell and incubated at 37°C. Percentage of transgeneexpressing cells was determined by flow cytometry 48 h post-transduction.In vitro LY6A surface binding assays and Transduction Assays

[0134] For all LY6A experiments, HEK293T cells were seeded in 15 cm dishes. 24 h later, cells were transfected with PEI MAX and 25 pg of Ly6a cDNA. 48 h later, cells were transferred into 24-well format at a density of 4 x 105 / well and transduced with respective AAV variants at a MOT of 10,000 vg per cell. For the surface binding assay, cells were prechilled to 4°C for 45 min and then the media was exchanged with fresh cold media containing the indicated recombinant AAV (10,000 vg per cell). 1 h later, cells were washed three times with cold PBS, then lysed for genomic DNA extraction and ddPCR analyses.

[0135] For transduction assays, AAV variants were added to cells (2 x 105 / well) and incubated at 37°C. 48 h post-transduction cells were harvested, and percentage of transgeneexpressing cells was determined by flow cytometry.

[0136] For immunofluorescence, HEK293T cells were seeded on poly-D-lysine-coated sterile glass coverslips (High Precision, Cat# 0117530) and incubated with respective AAV for 48 h post-transfection in 12-well plates. Cells were then fixed in 4% PFA for 10 min at room temperature. Coverslips were permeabilized with 0.1% PBS-T then blocked with PBS-T containing 10% normal goat serum for 30 min and incubated in primary antibody LY6A (1 :250 dilution; Invitrogen, 14-5981-82) diluted in blocking buffer overnight at 4°C. Coverslips were washed three times in PBS and then incubated with goat anti-rat Alexa Fluor Plus 647 secondary antibody (1 : 500, Thermo Fisher, A21247) in l x PBS for 90 min at room temperature followed by DAPI at 1 : 1,000 in PBS. Coverslips were then mounted on slides. Once dry, fluorescent microscopy images were captured on a Leica Stellaris 8 confocal microscope using a 63x objective. Acquired images were then processed using Image! software.Statistical analysis

[0137] Statistical significance was assessed using GraphPad Prism 9 software. The test used is specified in figure legends. For all statistical analyses, significance is represented as *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001; not significant, p>0.05.Table 2: Primer and probe sequencesExample 2. In vivo selection of shuffle capsids results in enrichment of capsids closely related to AAV clade A

[0138] A shuffled AAV capsid library based on parental variants AAV1 to AAV12 was used to produce and identify AAV capsid with improved specificity and transduction efficiency in theCNS. In addition to potential identification of highly functional novel AAV variants, this type of library can also enable identification of key capsid signatures, or fragments, important for CNS transduction.

[0139] The capsid library was cloned into the functional transduction (FT)-library platform and packaged into AAV particles for downstream assessment of capsid performance. The packaged AAV library was quantified using ddPCR and was injected into male naive Fah“ / “ / Rag2“ / “ / II2rg_ / “ (FRG) mice at a dose of 5 x 1011vector genomes (vg) / animal using i.v. injection. The brain was harvested three weeks post-injection and genomic DNA was extracted. Following PCR-based recovery of the enriched capsid sequences, the PCR amplicon was used to clone the library into the naive FT recipient backbone to generate the secondary library. In total, four consecutive rounds of selection were performed to allow for sufficient enrichment of AAV variants with enhanced functional transduction ability in the CNS. Following the fourth round of selection, the capsid library was amplified from DNA and full-length capsid sequences were obtained from N = 37 random clones. The sequence analysis revealed enrichment of a unique subpopulation of capsids closely related to the Clade A AAV serotype family. Subsequently, N = 16 capsid variants (referred to as AAV-BBB variants) were selected for further evaluation based on diverse representation across the phylogram.

[0140] To perform functional evaluation of the selected capsids, each capsid coding sequence was cloned into a standard AAV packaging plasmid downstream of the AAV2 rep gene, with AAV9 capsid additionally included for comparison. Each construct was subsequently used to package barcoded single stranded (ss)AAV-hSyn-mCherry-BC-WPRE-BGHpA genomes using a standard iodixanol purification protocol. The barcode (BC) region allows for the identification and quantification of each capsid, facilitating robust parallel analysis of capsid performance to be assessed using next generation sequencing (NGS). The use of two unique BCs per variants serves as an internal control.

[0141] Quantification analysis indicated that 5 of the selected AAV variants were inefficient at packaging and were thus excluded from further analysis. The remaining 11 variants (AAV-BBB3, AAV-BBB5, AAV-BBB6, AAV-BBB11, AAV-BBB27, AAV-BBB28, AAV-BBB31, AAV- BBB37, AAV-BBB38, AAV-BBB50 and AAV-BBB52; capsid sequences set forth in Table 4) were subsequently evaluated for their ability to transduce the CNS following i.v. administration in naive FRG mice. To enable direct side-by-side comparison, the 11 AAVs were mixed at an equimolar ratio to create an 'AAV-BBB Testing Kit' similar to the strategy previously published by Westhaus et al. (Hum Gene Ther. 2020;31(9-10):575-589) and injected i.v. at a total dose of 1.8 x 1011vg / animal (equivalent to 1.5 x 1010vg / variant) into two males FRG mice. Twenty-eight days post injection, the animals were sacrificed, with the brain and spinal cords harvested for analysis. Following DNA and RNA extraction and cDNA synthesis, the resulting nucleic acid was sent for NGS analysis to determine capsid performance at the cell entry (DNA) and expression (RNA) levels.

[0142] The analysis of the NGS reads revealed that 10 out of 11 assessed variants (all except AAV-BBB37) were able to transduce both the brain and the spinal cord at a significantly higher efficiency than the control AAV9, at both the DNA and RNA levels (Figure 1). Among the assessed variants, AAV-BBB6, AAV-BBB28, and AAV-BBB31 emerged as the top performers, exhibiting a remarkable >270-fold and >500-fold improvement over AAV9 at the RNA level in the brain and spinal cord, respectively (Figure 1C and D). Consequently, these three vectors were selected for further analysis, including comprehensive characterisation of vector efficiency in the mouse CNS, as well as analysis of their abilities to target peripheral tissues.Example 3. AAV-BBB28 and AAV-BBB31 demonstrate CNS transduction in mice with significant liver de-targeting when compared to AAV9 and AAV1

[0143] Before conducting the in vivo evaluation, analysis of vector packaging efficiency of AAV-BBB6, AAV-BBB28, and AAV-BBB31 in comparison to AAV9 was performed. While the packaging efficiency of AAV-BBB28 was lower than AAV9, two new variants AAV-BBB6 and AAV- BBB31 packaged with similar efficiency to AAV9.

[0144] Detailed sequence analysis revealed that AAV-BBB6, AAV-BBB28 and AAV-BBB31 differed from their closest parental serotype, AAV1, by only 12, 20 and 22 amino acids respectively, corresponding to a sequence identity of 95.5%, 97.55% and 97.28%, respectively, with AAV1 (see alignment in Figure 2). Modelling of the VP3 monomers of the AAV-BBB variants revealed structural difference only evident within the surface exposed VR-V loop when compared to AAV1 (data not shown). Interestingly, there were only two mutations within the VR-V region which were present in all three AAV-BBB variants: deletion of the asparagine (N) at position 498 (N498) relative to AAV1, and a threonine to alanine substitution at position 502 (T502A) relative to AAV1 (see Figure 2). Given this information, it was hypothesised that these two mutations were responsible for the observed structural change in VR-V and that they likely had significant impact on function of the AAV-BBB variants given their conservation across the top-performing AAV-BBB variants. Interestingly, it has been previously shown that T502 is an important residue for AAV1 interaction with sialic acid (Huang et al. J Virol. 2016;90(ll):5219-5230) and A502 is a required AAV9 residue for both galactose binding and post-attachment viral processing (Adachi et al. Nat. Commun 2014; 5, 3075. To determine if the two changes seen in the AAV-BBB variants affected their ability to bind sialic acid, two types of Chinese hamster ovary (CHO) cell lines were used: Pro5 and Lec2. Specifically, Pro5 lines contain surface exposed sialic acid residues, whereas Lec2 cells have exposed surface glycans due to mutation in their sialic acid transporter. Due to the surface exposed sialic acid residues, CHO-Pro5 line facilitates the binding of AAVs that rely on sialic acid binding, such as AAV1, and CHO-Lec2 line facilitates the binding of galactose residue binding vectors, such as AAV9. Interestingly, all three AAV-BBB variants showed the same reduced level of cell binding and transgene expression in the Lec2 and Pro5 cells, as measured by the number of vector genomes bound to the cell (data not shown), and at the percentage of GFP positive cells quantified via FACS (data not shown). This finding suggests that the specific capsid changes present in the AAV-BBB variants abolished the binding to sialic acid and did notfacilitate binding to terminal galactose. This suggests that an alternative receptor is used by the AAV-BBB variants to bind to and enter the target cells.

[0145] To assess the individual performance of the novel AAV-BBB variants in comparison to AAV9 and AAV1 across various murine tissues, the variant capsids were used to package a ssAAV cassette encoding an mCherry fluorescent protein under the control of the cytomegalovirus (CMV) promoter (ssAAV-CMV-mCherry-WPRE-BGHpA). The ubiquitous CMV promoter was chosen in contrast to the previously used hSyn promoter to allow evaluation of the expression of the novel variants in CNS and other tissues.

[0146] Following vector production, the brain tropisms of the novel AAV-BBB variants were evaluated in adult male FRG mice (C57BL / 6J background). Individual variants were injected into mice at a dose of 5 x 1011vg / animal, with N = 3 animals per vector. Mice were harvested three weeks post-injection with their tissues harvested for further analysis of AAV efficiency, tropism and biodistribution (including brain, spinal cord, liver, heart, muscle and kidney).

[0147] Compared to AAV9 and AAV1, a significant increase in vector copy number (VCN) in the mouse brain for AAV-BBB28 and AAV-BBB31 was observed, with a 49-fold and 55-fold increase, respectively (Figures 3A and 3B). Although not as pronounced, noticeable increases in VCN were also observed in the spinal cord for all variants. Notably, all vectors exhibited significant de-targeting from murine liver, with the liver VCN of AAV-BBB6, AAV-BBB28, and AAV-BBB31 measured to be 224-fold, 162-fold, and 27-fold lower than AAV9, respectively (Figure 3C). Interestingly, the significantly lower liver transduction from the AAV-BBB variants differ from the parental AAV1 capsid as measured with VCN (Figure 3C).

[0148] The trends observed for the VCN aligned with the pattern of mCherry expression throughout the brain, spinal cord and livers of the treated mice (immunohistochemical data not shown). In the brain and spinal cord, the greatest expression was observed for AAV-BBB31, followed by AAV-BBB28 and AAV-BBB6. The regions with the highest tropism appeared to be the brainstem, cerebellum, midbrain and thalamus, with quantitative analysis of mCherry-positive neurons revealed a significant increase across all regions with each AAV-BBB variant compared to AAV9, except for the cortex and hippocampus (Figure 4A). Expression from the AAV-BBB variants was also observed in the spinal cord (Figure 4B).

[0149] To further elucidate the cellular tropism of the AAV-BBB variants, the colocalisation of vector-encoded mCherry with GFAP (astrocytes) and olig2 (oligodendrocytes) was examined and revealed minimal overlap (data not shown). In contrast, the AAV-BBB variants exhibited enhanced targeting of Purkinje cells in the cerebellum, with AAV-BBB28 demonstrating a significant increase in mCherry-positive Purkinje cells compared to AAV9 (data not shown). These findings collectively support the neuronal preference of the AAV-BBB variants, as indicated by mCherry co-localisation with NeuN and Calbindin.

[0150] To assess the biodistribution of the vectors in other peripheral tissues, VCN and mCherry expression in the heart, muscle, and kidneys was assessed. In the heart and muscle, nosignificant differences in VCN or mCherry fluorescence intensity were observed when compared to AAV9. Upon VCN analysis in the kidney, a significant increase in vector DNA for all AAV-BBB6 and AAV-BBB31 variants compared to AAV9 was observed (data not shown). However, the expression of the mCherry reporter at the protein level for all the AAV-BBB variants was virtually undetectable in the kidney. This result suggests that all three AAV-BBB variants can enter the kidney with high efficiency but are unable to functionally express their encoded transgene. This discrepancy between AAV entry and transgene expression in the kidney indicates incomplete intracellular trafficking.

[0151] In order to more closely evaluate the function of the AAV-BBB vectors in the CNS, AAV-BBB28, AAV-BBB31, and AAV9 were used to package a reporter cassette encoding mCherry reporter under the control of a neuron-restrictive hSyn promoter. The vectors were injected systemically (i.v.) into male naive FRG mice at a dose of 5 x lOnvg / animal (N = 2 animals per vector) and harvested three weeks post-injection. While the AAV-BBB variants continued to exhibit a preference for neuronal transduction using the ubiquitous CMV promoter, the switch to the hSyn promoter facilitated a more widespread transduction, leading to the observation of transduced neurons throughout the cortex (Figure 5). This expanded transduction pattern was not previously observed with the CMV promoter (microscopy images not shown). Quantification of the number of mCherry positive cells colocalised with NeuN in the cortex supported this observation, increasing significantly with the use of the hSyn promoter (2.5-fold and 4-fold improvement over the CMV promoter for AAV-BBB28 and AAV-BBB31, respectively) (Figure 5).Example 4. AAV-BBB variants demonstrate reduced targeting of human hepatocytes in a hFRG chimeric liver in vivo model compared to AAV9

[0152] Although the VCN and mCherry expression data confirm that our novel variants were significantly de-targeted from the mouse liver, it was important to study if the same effect would be observed for human liver as vector tropism can be inconsistent across species. Therefore, to determine whether our novel variants were also de-targeted from human primary hepatocytes, female FRG mice were engrafted with primary human hepatocytes to generate a well-established in vivo xenograft model of human liver. Humanised FRG (hFRG) mice were individually injected with the top two performing variants, AAV-BBB28 and AAV-BBB31, encoding ssAAV-CMV-eGFP transgene cassettes. AAV9 and AAV1 encoding the same cassette were included as controls. Mice were injected i.v. at a dose of 5 x 1011vg / animal (N = 2 animals per vector) and harvested 3 weeks post vector administration. The mouse brain and spinal cord were harvested for immunohistology and VCN analysis. For the chimeric liver, one lobe was isolated for immunohistology analysis prior to liver perfusion. The remaining liver was used to isolate the human hepatocyte population using Fluorescence Activated Cell Sorting (FACS). The isolated human hepatocytes were subsequently used for VCN analysis.

[0153] Consistent with the results obtained in the non-engrafted naive male FRG mice, AAV-BBB28 and AAV-BBB31 demonstrated a visible reduction in eGFP expression in the chimeric liver when compared to both AAV9 and parental AAV1 (Immunohistochemistry images notshown). For the AAV-BBB variants, the low level of eGFP expression present appeared to primarily co-localise with the mouse hepatocytes, as evident by the fact that eGFP positive cells appeared to be negative for the human GAPDH marker. This conclusion was further supported by the significant reduction in the percentage of eGFP-positive cells per human cluster (Figure 6A). VCN analysis revealed that while AAV-BBB31 has lower average VCN per human diploid genome, the difference was not statistically significant compared to AAV9. On the other hand, AAV-BBB28 showed a significantly lower average VCN in human cells (Figure 6B), which is consistent with the percentage of GFP positive human cells (Figure 6A), as compared with AAV9. Importantly, AAV1 also displayed a significantly lower human liver transduction compared to AAV9, suggesting that AAV-BBB28 and AAV-BBB31 retain the naturally reduced liver tropism properties observed in the parental AAV1 capsid.

[0154] Consistent with data obtained in the naive FRGs, AAV-BBB28 and AAV-BBB31 demonstrated stronger expression than AAV9 in the brain and the spinal cord. However, the VCNs were reduced compared to the non-engrafted male FRG mice receiving the same total vector dose i.v. (Figure 7). These differences may be attributed to the previously observed discrepancies in transduction efficiency between female and male C57BL76J mice. Another possible explanation is that the higher VCN in the hFRG liver compared to the naive FRG liver may result in partial sequestration of the AAV, reducing the amount of free circulating AAVs in the blood available for BBB crossing. It is also possible that the observed effect is a net result of both mechanisms.Example 5. Rational refinement of antigenic footprints results in improved IVIg resistance

[0155] As pre-existing immunity against AAV remains one of the major challenges in clinical gene therapy application, the AAV-BBB variants were assessed for their ability to resist neutralisation by human pooled intravenous immunoglobulin (IVIg). To do so, an in vitro AAV neutralisation assay was performed on HEK293T cells at increasing concentrations of IVIg, using AAV9 as a control (Figures 8A-8C). Notably, the results revealed that the AAV-BBB variants exhibited decreased resistance to IVIg neutralization when compared to AAV9 (Figure 8A). The data showed that the concentration of IVIg required to achieve 50% decrease in transduction (IC50) was lower for all AAV-BBB variants in comparison to AAV9 (~l / 8 for AAV-BBB variants vs 1 / 4 for AAV9) (Figure 8A). This finding correlates to a lower concentration of IVIg required to inhibit cellular transduction of our AAV-BBB variants compared to AAV9.

[0156] To bypass the immune system to retain the safety and efficacy of AAV vectors, Tse et al. mapped key regions on AAV1 that are responsible for capsid interaction with the Nabs (Tse et al. Proc National Acad Sci. 2017;114(24):E4812-E4821). As the AAV-BBB variants are highly homologous to AAV1, it was hypothesised that by altering the capsids antigenic footprints, the recognition by the immune system could be bypassed while retaining the functional properties of our AAV vectors. To this end, the two key antigenic sites identified by Tse et al. were modified in the AAV-BBB variants with the aim to increase their immune escape properties. Specifically, amino acid residues within two surface regions— VR-IV (456-AQNK-459), and VR-VIII (588-STDPATGDVH-597) were replaced with (456-SERR-459) and (588-DLDPKATEVE-597), respectively.

[0157] In the case of AAV-BBB28, the addition of the VR-IV or VR-VIII mutation improved the resistance to the IVIg, with the inclusion of the VR-VIII mutation having the most effect (Figure 8B). Interestingly, in the context of AAV-BBB31, the VR-IV mutation had no effect on IVIg resistance while the VR-VIII mutation resulted in a decreased resistance to IVIg neutralization compared to AAV-BBB31 (Figure 8C). The prevalence of NAbs against the AAV-BBB variants was subsequently analysed using a small cohort of individual human sera (N = 8) but no significant differences in seroprevalence between the AAV-BBB variants and control AAV9 was found (data now shown). Interestingly, while AAV-BBB28. VR-IV showed an increased ability to escape NAbs (Figure 8B), the VR mutant displayed a seroprevalence comparable to AAV9, and AAV-BBB28 (data not shown).

[0158] To assess whether the immune escape variants of AAV-BBB28 retained the ability to cross the BBB in vivo following the capsid modifications, AAV-BBB28, AAV-BBB28. VR-IV and AAV-BBB28. VR-VIII variants were injected i.v. at a dose of 2 x 1011vg / animal into naive male FRG mice. Although analysis of VCN in the brain indicated no significant decrease in vector's ability to cross the BBB between AAV-BBB28 and AAV-BBB28. VR-IV, the ability of AAV-BBB28. VR-VIII to cross the BBB appeared to be abolished (Figures 8D and E). Despite the capsid modification having no impact on the ability of AAV-BBB28. VR-IV to enter cells, IHC analysis indicated a reduction in transgene expression from the AAV-BBB28. VR-IV variant when compared to AAV- BBB28 (data now shown). This reduction in expression was particularly noticeable when observing the number of GFP+ cells in the midbrain, hippocampus and cortex region (data now shown).

[0159] Preliminary assessment of AAV-BBB6 with its AAV-BBB6. VR-IV and AAV- BBB6. VR-VIII variants revealed the same trend as AAV-BBB28, showing an increase in immune escape, although resulting in a reduction of brain transduction with AAV-BBB6. VR-IV and a complete impairment of BBB crossing for AAV-BBB6. VR-VIII (Figures 9A-9C).Example 6. Novel AAV-BBB variants fail to cross the BBB in BALB / cJ mice due to LY6A dependency

[0160] Previous published studies reported that AAV's ability to traverse the BBB may differ between mouse strains. One such example is AAV9 variant, PHP.B, which is able to traverse the BBB in C57BL / 6J mice but not BALB / cJ (see.e.g. Hordeaux et al. Mol Ther. 2019;27(5) :912- 921).

[0161] To evaluate the BBB crossing efficiency of the AAV-BBB variants in BALB / cJ mice, AAV-BBB28 and AAV9 as a control were injected i.v. into BALB / cJ mice. Although improvement in IVIg escape could not be achieved previously with AAV-BBB31, due to its superior transduction ability, it was also included in this analysis. Each variant was administered at a dose of 5 x 1011vg / animal, and brain, spinal cord, and liver tissues were collected for analysis three weeks post- injection.

[0162] Analysis revealed no detectable transgene expression or VCN in the brain or spinal cord of mice injected with the AAV-BBB variants (data now shown). While the ability to cross the BBB was not maintained in BALB / cJ mice, the observed detargeting effect in the liver, previously observed in FRG mice, remained consistent (data not shown).

[0163] When it was previously observed that the AAV-PHP.B family of vectors were unable to cross the BBB in BALB / cJ mice, further investigations revealed that they exhibited a dependency on the LY6A receptor for transport into the CNS. Notably, LY6A is a receptor that is unique to certain mouse strains and most critically is not present in NHPs or humans. To investigate whether the AAV-BBB variants also utilise LY6A for BBB binding and crossing, an in vitro binding assay and expression analysis using HEK293T cells, with and without Ly6a expression Ly6a and -, respectively) was performed. The results showed that the binding capacity of AAV-BBB28, and AAV-BBB31 increased by more than 100-fold in Ly6a expressing cells, similarly to what was reported for AAV-PHP.eB (Figure IDA). This trend was further supported by expression analysis performed by FACS, which demonstrated a significant increase in transduction efficiency (Figure 10B). Similarly, the GFP mean fluorescence intensity (MFI) was significantly higher for the AAV-BBB variants in the presence of Ly6a (Figure IOC). Importantly, no significant changes in binding or transduction ability were observed for AAV9, regardless of Ly6a expression.

[0164] These findings show that the enhanced binding and transduction capacity of AAV- BBB28 and AAV-BBB31 variants in the presence of LY6A are consistent with the behaviour reported for AAV-PHP.eB, indicating a shared dependency on the LY6A receptor as a gateway to the brain.

[0165] Interestingly, the LY6A receptor is also utilised for other well-known AAV9 variants, including AAV. CAP-BIO and AAV.CAP-B22, despite the different bioengineering methods used to develop those vectors. AAV-PHP.eB is an AAV9 variant which include peptide additions in VR-VIII, and AAV.CAP-B10 has the same structure as AAV-PHP.eB with the addition of a peptide substitution in VR-IV. The insertion in the VR-VIII loop is known to be responsible for facilitating LY6A binding in these AAV9 variants. In contrast, AAV-BBB variants described herein were selected from a shuffled capsid library using FT selection method and display the highest homology to AAV1, and does not have the VR-VIII loop insertion.

[0166] Although the LY6A receptor is not present in BALB / cJ mice, non-human primates or humans, studies have demonstrated that AAV.CAP-B10 and AAV.CAP-B22 exhibit significantly enhanced efficiency in crossing the BBB in marmosets compared to AAV9, despite their inability to cross the BBB in BALB / cJ mice. AAV.CAP-B22 also shows comparable BBB crossing and CNS transduction capabilities to AAV9 in macaques, a species of NHP more closely related to humans. As such, it is expected that the AAV-BBB vectors described herein would also display comparable BBB crossing and CNS transduction capabilities in non-human primates and humans.

[0167] Additionally, delivery of these vectors through intra-CSF routes, such as IT injection, holds promise. IT injection could offer even greater specificity to the CNS with a reducedoverall dose. Notably, the AAV-BBB variants described herein share close similarities with AAV1, which has demonstrated high efficiency in CNS transduction following IT injection in NHP models. Additionally, the LY6A-binding variant, AAV-PHPe.B, has shown excellent performance following IT delivery in NHP models, albeit with a predominantly astrocytic tropism. In contrast, the AAV- BBB variants of the present disclosure exhibit neuronal targeting, suggesting the potential for high efficiency in transducing neurons following IT injection. While IT injection of AAV9 in NHP models has resulted in vector leakage into the periphery, leading to strong liver transduction, due to the enhanced liver-detargeting achieved in both murine and human hepatocytes with the AAV- BBB variants, the off-target effects resulting from IT leakage is expected to be minimal.Example 6. Identification of residues important for crossing BBB

[0168] Further studies were performed to identify the regions responsible or important for the AAV-BBB variants' ability to cross the BBB. Alignment of AAV1 and AAV-BBB6 indicated that there were three key regions with variations between the two capsids that were likely involved in BBB crossing: Region 1: residues at positions 275 to 351; Region 2: residues at positions 352 to 441; and Region 3: residues at positions 442-532 (numbering relative to the AAV1 capsid set forth in SEQ ID NO: 1). Region 3 included the N498del and T502A mutations in AAV-BBB6 compared to AAV1.

[0169] AAV1 variants (C1-C6) containing regions 1, 2 and / or 3 from AAV-BBB6 were generated (Table 3; where 0 is no region, and 1 is 1 region) and injected i.v. at a total dose of 1.8 x 1012vg / animal (equivalent to 1.25 x 1011vg / variant) into two non-engrafted FRG mice and the transduction of the brain was assessed by NGS at the DNA level. As shown in Figure 11, AAV with capsid polypeptides that contained region 3 of AAV-BBB6 had the ability to cross the BBB and transduce cells in the brain.Table 3

[0170] AAV1 variants comprising the N498del or T502A mutations were then generated and also assessed, as were AAV-BBB6 variants with the reverse mutations (i.e. A501T and 498insN), and compared to parental strain AAV1 and the AAV1RX variant which contains keyresidues from RhlO (VR1 region) and can cross the BBB (Albright et al. Mol Ther. 2018;26(2) :510- 523). As shown in Figure 12, while AAV-BBB6 variants with A501T or 498insN had significantly reduced brain transduction compared to AAV-BBB6, AAV1 variants comprising the N498del or T502A mutations did not exhibit significant BBB crossing and transduction.

[0171] An AAV1 variant containing both the N498del and T502A mutations was generated and further studies were performed, where the various capsid variants were i.v. administered into male FRG mice (N = 2, dose: 5 x 1011total vg / animal). As shown in Figure 12, this AAV1-N498del- T502A variant was able to cross the BBB and transduce cells in the brains to levels similar to that of AAVB6. This data suggested that both mutations are required for efficient BBB crossing.

[0172] To further examine the importance of the N498del and T502A mutations, variants of AAV-BBB28, AAV-BBB31 and AAV-BBB37 were produced: AAV-BBB28-A502T, AAV-BBB28- 499insN, AAV-BBB31-A502T, AAV-BBB31-499insN, AAV-BBB37-T502A, AAV-BBB37- N498del. The various capsid variants were i.v. administered into male FRG mice (N = 2, dose: 1 x 1011total vg / animal). As shown in Figure 13, while reversal of the N498del and T502A mutations in AAV- BBB28 and AAV-BBB31 capsids abrogated BBB crossing, introduction of these mutations in AAV- BBB37 did not completely facilitate BBB crossing.Table 4. Capsid Sequences

Claims

CLAIMS:

1. An AAV capsid polypeptide, wherein the capsid polypeptide: a) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3-13; b) comprises an amino acid sequence having at least 96%, 97%, 98% or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 3-13; c) comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity to the sequence set forth in any one of SEQ ID NOs: 3-13 and comprises one or more modifications relative to SEQ ID NO: 1, wherein the one or more modifications are selected from among D4G, Y6C, N14T, E21Q, D24A, D24K, A29P, A29V, K31Q, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, D41N, G42S, G42R, F56G, A67E, R92K, Q105K, V125L, L129F, G135A, G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, D178E, S179T, P185D, L188I, S205A, A263Q, T265del, S268del, T326Q, V330T, S345T, I373V, I373N, N383D, T411Q, T415E, E418N, E418D, N496K, N497Q, N498del, S499Q, N500Q, F501L, T502A, F584L, A598V, A601V, H628N, L640M, N642H, V699I, A706Y, A709T, N710S, T714A, D716N, N717T, N718E, L720V, T722S and P735N ; or d) is a VP2 or VP3 fragment of any one of a) - c), wherein the VP2 fragment comprises residues at the positions corresponding to positions 138-736 of the AAV1 polypeptide set forth in SEQ ID NO: 1 and wherein the VP3 fragment comprises residues at the positions corresponding to positions 204-736 of the AAV1 polypeptide set forth in SEQ ID NO: 1.

2. The AAV capsid polypeptide of claim 1, comprising one or more modifications selected from among D24A, A29V, K31Q, K38H, D41N, G42R, L129F, G135A, G141A, Q148P, 151- 152insR, E152S, S157T, T162K, K168R, D178E, P185D, S205A, S268del, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N496K, N497Q, N498del, S499Q, N500Q, F501L, T502A, F584L, A598V, A601V, H628N, L640M, N642H and V699I.

3. The AAV capsid polypeptide of claim 1 or 2, comprising the mutation N498del and / or the mutation T502A relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

4. An AAV capsid polypeptide, wherein the capsid polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity to the sequence set forth in any one of SEQ ID NOs: 3-9 or 11-13, or a VP2 or VP3 fragment thereof, wherein: the VP2 fragment comprises residues at the positions corresponding to positions 138-736 of the AAV1 polypeptide set forth in SEQ ID NO: 1 and wherein the VP3 fragment comprises residues at the positions corresponding to positions 204-736 of the AAV1 polypeptide set forth in SEQ ID NO: 1; and the capsid polypeptide comprises the modifications N498del and T502A relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

5. The AAV capsid polypeptide of claim 4, further comprising one or more modifications selected from among D4G, Y6C, N14T, E21Q, D24A, D24K, A29P, A29V, K31Q, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, D41N, G42S, G42R, F56G, A67E, R92K, Q105K, V125L, L129F, G135A, G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, D178E, S179T, P185D, L188I, S205A, A263Q, T265del, S268del, T326Q, V330T, S345T, I373V, I373N, N383D, T411Q, T415E, E418N, E418D, N496K, N497Q, N498del, S499Q, N500Q, F501L, T502A, F584L, A598V, A601V, H628N, L640M, N642H, V699I, A706Y, A709T, N710S, T714A, D716N, N717T, N718E, L720V, T722S and P735N.

6. The AAV capsid polypeptide of claim 4 or 5, comprising one or more modifications selected from among D24A, A29V, K31Q, K38H, D41N, G42R, L129F, G135A, G141A, Q148P, 151- 152insR, E152S, S157T, T162K, K168R, D178E, P185D, S205A, S268del, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N497Q, S499Q, N500Q, F501L, F584L, A598V, A601V, H628N, L640M, N642H and V699I relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

7. The AAV capsid polypeptide of any one of claims 1-6, comprising one or more modifications selected from among T326Q, V330T, S345T, N383D, T411Q, T415E and E418N relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

8. The AAV capsid polypeptide of claim 7, comprising modifications T326Q, V330T, S345T, N383D, T411Q, T415E and E418N relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

9. The AAV capsid polypeptide of any one of claims 1-8, comprising one or more modifications selected from among from D24A, K31Q and D178E relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

10. The AAV capsid polypeptide of claim 9, comprising modifications D24A, K31Q, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del and T502A, relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

11. The AAV capsid polypeptide of any one of claims 1-8, comprising one or more modifications selected from G141A, Q148P, 151-152insR, E152S, S157T, T162K, K168R, P185D, S205A, N642H and V699I relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

12. The AAV capsid polypeptide of claim 11, comprising modifications G141A, Q148P, 151- 152insR, E152S, S157T, T162K, K168R, P185D, S205A, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, 642H and V699I relative to the AAV1 capsid polypeptide set forth in SEQ ID NO:

113. The AAV capsid polypeptide of any one of claims 1-8, comprising one or more modifications selected from D24A, G135A, Q148P, 151-152insR, E152S, S157T, T162K, D178E, A598V, A601V, H628N, L640M and N642H relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

14. The AAV capsid polypeptide of claim 13, comprising the modifications D24A, G135A, Q148P, 151-152insR, E152S, S157T, T162K, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E,E418N, N498del, T502A, A598V, A601V, H628N, L640M and N642H relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

15. The AAV capsid polypeptide of any one of claims 1-8, comprising one or more modifications selected from D24A, A29V, K31Q, K38H, D41N, G42R, L129F, Q148P, 151-152insR, E152S, S157T, T162K, D178E, N496K, N497Q, S499Q, N500Q, F501L, F584L, A598V and N642H relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

16. The AAV capsid polypeptide of claim 15, comprising the modifications D24A, A29V, K31Q, K38H, D41N, G42R, L129F, Q148P, 151-152insR, E152S, S157T, T162K, D178E, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N496K, N497Q, N498del, S499Q, N500Q, F501L, T502A, F584L, A598V and N642H relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

17. The AAV capsid polypeptide of any one of claims 1-8, comprising one or more modifications selected from D24A, Q148P, 151-152insR, E152S, S157T, T162K, D178E, S268del, F584L and A598V relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

18. The AAV capsid polypeptide of claim 17, comprising the modifications D24A, Q148P, 151- 152insR, E152S, S157T, T162K, D178E, S268del, T326Q, V330T, S345T, N383D, T411Q, T415E, E418N, N498del, T502A, F584L and A598V relative to the AAV1 capsid polypeptide set forth in SEQ ID NO: 1.

19. The AAV capsid polypeptide of any one of claims 1-18, comprising a modification that confers increased immune evasion on an AAV vector comprising the capsid polypeptide.

20. The AAV capsid polypeptide of claim 19, wherein the modification is replacement of 456- AQNK-459 with 456-SERR-459.

21. The AAV capsid polypeptide of claim 19, wherein the modification is replacement of 588- STDPATGDVH-597 with 588-DLDPKATEVE-597.

22. An AAV vector, comprising the capsid polypeptide of any one of claims 1 to 21.

23. The AAV vector of claim 22, wherein the vector exhibits decreased transduction efficiency of a cell of the liver, heart, kidney or muscle compared to an AAV1 or AAV9 vector comprising a capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2.

24. The AAV vector of claim 22 or 23, wherein the vector exhibits decreased transduction efficiency of a hepatocyte compared to an AAV9 vector comprising a capsid polypeptide comprising the amino acid sequence of SEQ ID NO:2.

25. The AAV vector of any one of claims 22 to 24, wherein transduction efficiency is decreased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

26. The AAV vector of any one of claims 22-25, wherein the vector exhibits increased in vivo transduction efficiency of a cell of the central nervous system compared to an AAV1 or AAV9 vector comprising a capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2.

27. The AAV vector of claim 26, wherein the cell of the central nervous system is in the brainstem, cerebellum, midbrain, thalamus, spinal cord, cortex or hippocampus.

28. The AAV vector of claim 26 or 27, wherein transduction efficiency is increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or 500%.

29. The AAV vector of any one of claims 22 to 28, further comprising a heterologous coding sequence.

30. The AAV vector of claim 29, wherein the heterologous coding sequence encodes a peptide, polypeptide or polynucleotide.

31. The AAV vector of claim 30, wherein peptide, polypeptide or polynucleotide is a therapeutic peptide, polypeptide or polynucleotide.

32. An isolated nucleic acid molecule encoding the capsid polypeptide of any one of claims 1 to 21.

33. A vector comprising the nucleic acid molecule of claim 32.

34. The vector of claim 33, wherein the vector is selected from among a plasmid, cosmid, phage and transposon.

35. A host cell, comprising the AAV vector of any one of claims 22 to 31, the nucleic acid molecule of claim 32, or the vector of claim 33 or claim 34.

36. A method for introducing a heterologous coding sequence into a host cell, comprising contacting a host cell with the AAV vector of any one of claims 29 to 31.

37. The method of claim 36, wherein the host cell is a cell of the central nervous system.

38. The method of any claim 36 or 37, wherein contacting the host cell with the AAV vector comprises administering the AAV vector to a subject.

39. The method of claim 38, wherein administration of the AAV vector to the subject effects treatment of a disease or condition of the central nervous system.

40. The method of claim 36 or 37, wherein the method is performed in vitro or ex vivo.

41. Use of the AAV vector of any one of claims 22 to 31 for the preparation of a medicament for treating a disease or condition of the central nervous system.

42. A method for producing an AAV vector, comprising culturing a host cell comprising a nucleic acid molecule encoding the capsid polypeptide of any one of claims 1 to 21, an AAV rep gene, a heterologous coding sequence flanked by AAV inverted terminal repeats, and helper functions for generating a productive AAV infection, under conditions suitable to facilitate assembly of an AAV vector comprising a capsid comprising the capsid polypeptide of any one of claims 1 to 21, wherein the capsid encapsidates the heterologous coding sequence.

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