EP4680619A1

EP4680619A1 - Engineered adeno-associated viruses

Info

Publication number: EP4680619A1
Application number: EP24771303.5A
Authority: EP
Inventors: Mohamed Amine MELIANI; Yu Fen Samantha SEAH; Anna TRACZYK; Wei Leong CHEW
Original assignee: Agency for Science Technology and Research Singapore
Current assignee: Agency for Science Technology and Research Singapore
Priority date: 2023-03-13
Filing date: 2024-03-13
Publication date: 2026-01-21
Also published as: CN121039150A; WO2024191354A1

Abstract

Disclosed herein are compositions comprising at least one modified adeno-associated viral (AAV) capsid protein. Also disclosed herein are adeno-associated virus particles comprising the AAV capsid proteins disclosed herein. In one example, the compositions, nucleic acids, vectors, host cells and adeno- associated virus particles disclosed herein are used in therapy.

Description

ENGINEERED ADENO-ASSOCIATED VIRUSES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 J This application claims the benefit of priority of Singapore provisional application no. 10202300688R, filed 13 March 2023, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of biotechnology. In particular, the present invention relates to engineering and/or attenuating adeno-associated viruses.

BACKGROUND OF THE INVENTION

[0003] Recombinant viral vectors based on adeno-associated virus (AAV) have emerged as an ideal delivery platform for in vivo gene transfer and genome editing therapies. AAV vectors are derived from wild-type viruses that naturally infect humans. As a consequence, depending on the AAV vector serotype, up to two-thirds of humans arc seropositive to AAV, thereby impacting the use of AAV for gene transfection and transfer. Since AAV capsid structures are very similar' with high amino acid sequence homology, the various AAV serotypes can encounter cross-reactivity to antibodies (i.e., naturally occurring AAV serotypes have conserved sequences that are neutralized by the same antibodies).

[0004] Thus, there is an unmet need for adeno-associated viruses that are capable of evading preexisting anti- AAV antibodies.

SUMMARY

[0005] In one aspect, the present disclosure refers to a composition comprising at least one modified adeno-associated viral (AAV) capsid protein, wherein the composition is resistant to neutralisation or is not neutralised by human neutralising antibodies, wherein the at least one modified adeno-associated viral (AAV) capsid protein is modified at any one or more of residues 24 to 47, 48 to 97, 72 to 121, 120 to 167 and 168 to 217 of an adeno-associated viral (AAV) VP1 capsid protein or an ortholog thereof.

[0006] In another aspect, the present disclosure refers to a nucleic acid sequence encoding the composition described herein, or encoding the one or more modified adeno-associated viral (AAV) capsid proteins described herein.

[0007] In yet another aspect, the present disclosure refers to a vector comprising the nucleic acid sequence disclosed herein.

[0008] In a further aspect, the present disclosure refers to a host cell comprising the vector described herein or the nucleic acid sequence described herein. [0009] In one aspect, the present disclosure refers to the composition disclosed herein, the nucleic acid disclosed herein, the vector disclosed herein, the host cell disclosed herein, or the adeno-associated virus particle disclosed herein, for use in gene therapy.

[0010] In yet another aspect, the present disclosure refers to a method of identifying an adeno- associated viral (AAV) capsid protein or peptide which is resistant to neutralisation or is not neutralised by human neutralising antibodies, the method comprising (a) contacting the adeno-associated viral capsid protein or peptide with sera known to contain adeno-associated virus neutralising antibodies, thereby allowing the neutralising antibodies to bind to an epitope comprised within the adeno-associated viral capsid protein or peptide; (b) removing the neutralising antibody-bound epitope from the adeno-associated viral capsid protein or peptide; and (c) identifying the adeno-associated viral capsid protein or peptide that did not bind to the neutralising antibodies.

[0011] In a further aspect, the present disclosure refers to a method of preventing an immune response to an engineered adeno-associated virus, the method comprising administering to a subject an engineered adeno-associated virus comprising at least one modified adeno-associated viral (AAV) capsid protein described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which: [0013] FIG. 1 shows a summary of phage immunoprecipitation sequencing method. FIG. 1 A shows an AAV and CRISPR-Cas phage library that were designed with protein sequences from UniProt and NCBI Protein database. The library consists of roughly 10 000 peptides tiles of 49 amino acids with 25 amino acid overlaps. The DNA sequences encoding the peptides tiles were synthesized, cloned, and packaged into T7 phages that display the encoded peptide on the phage surfaces. Next, the phage library was mixed and incubated with patient serum that contains antibodies. The antibodies bound to their target peptide sequences as displayed by the phages, and the complexes were enriched from the sample using protein A/G- coupled beads (IP - immunoprecipitation). After the immunoprecipitation, the genomes of phages bound to antibodies were amplified by polymerase chain reaction (PCR) and sequenced, identifying the peptide sequences bound by the antibodies. The results are represented by a heat map where darker shade represents higher level of antibodies recognizing particular epitope. Assessment of the library representation as an input library (FIG. IB) and as a cloned library (FIG. 1C), before and after cloning and packaging, are shown. FIG. ID shows the results of assessment of the library quality by Sanger sequencing of 20 randomly chosen clones.

[0014] FIG. 2 shows the heatmap results of epitope-mapping of AAV serotypes by human antibodies using PhTP-seq. AAV epitopes recognized by antibodies in pooled serum sample (left) or an individual patient’s scrum (right) arc shown. [0015] FIG. 3 shows a column graph showing AAV variants showing increase vector yield. Wild-type AAV-DJ and AAV variants packaging a self-complementary green fluorescent protein (GFP) genome were produced by triple-transfection in HEK293T cells and titrated by quantitative polymerase chain reaction (PCR). Results are shown as mean ± SD of two independent titrations.

[0016] FIG. 4 shows fluorescent microscope images of AAV variants showing superior transduction efficiency and neutralisation escape. FIG. 4A shows the results of in vitro transduction of HEK293 cells with AAV vectors encoding GFP at a multiplicity of infection (MOI) of 20 000. Analysis was performed 48 hours later. FIG. 4B shows the results of an in vitro neutralisation assay: WT AAV-DJ or AAV- DJ D213A encoding a gr een fluorescent protein (GFP) were mixed with a serial dilution of human sera for 1 hour at 37°C. The mixture was then added to the cells and incubated for 48 hours at 37°C.

[0017] FIG. 5 shows fluorescent microscope images of AAV variants showed escape from pre-existing NAb in human serum. Results of in vitro neutralisation assay using WT AAV2 versus AAV2 D213A (FIG. 5A), WT AAV9 versus AAV9_D213A (FIG. 5B) and WT AAD-DJ versus AAV-Dj_A70R (FIG. 5C). AAV vectors at a multiplicity of infection (MOI) of 50 000 (serotypes 2 and 9) or 20 000 (serotype DJ) were mixed with a serial dilution of human sera for 1 hour at 37°C. The mixture was then added to the cells and incubated for 48 hours.

[0018] FIG. 6 shows a line graph showing results indicating complete resistance to pre-existing neutralising antibodies in human serum with AAV-DJ_D213A and AAV-DJ_A70R_D213A. This figure illustrates the impact of either the D213A or the A70R_D213A amino acid substitutions on the neutralization by pre-existing neutralising antibodies (Nabs) in AAV-DJ. The experiment was performed using an in vitro neutralization assay. As a control for 100% transduction efficiency, vectors were mixed only with DMEM (no serum.) The mixture was incubated for 1 hour at 37°C before being added to the cells. After 48 hours of incubation, the green fluorescent protein (GFP) expression was measured. The dotted line at 50 represents neutralising antibody (Nab) -mediated inhibition of AAV transduction by 50%. Solid lines represent relative transduction efficiencies of WT AAV (black), AAV_D213A (Grey, dotted), and AAV-DJ_A70R_D213A (black, dotted) at different dilutions of scrum. Error bars represent SD (n = 2 for WT AAV-DJ and AAV-DJ_D213A and n=l for AAV-DJ_A70R_D213A).

DETAILED DESCRIPTION

[0019] Adeno-associated viruses (AAV) are commercially and clinically effective delivery vectors but are commonly neutralised and rendered ineffective by antibodies in the patients. Thus, the present disclosure describes compositions comprising AAV capsids that are resistant to, or capable of, evading neutralisation by neutralising antibodies that may be present in a host or subject.

[0020] As used herein, the term “AAV” or “adeno-associated virus” refers to a non-pathogenic, small viruses of the Parvovirus family which infect humans. The term “AAV” includes, but is not limited to, AAV subtypes, for example, AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), and AAV type 8 (AAV-8). Furthermore, as used herein, the terms “AAV virus”, “AAV viral particle”, or “rAAV vector particle” refer to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated nucleotide recombinant AAV vector. If the viral particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as a recombinant AAV vector (rAAV vector), a rAAV particle, or a rAAV vector.

[0021 ] Disclosed herein are compositions comprising AAV capsids resistant to neutralisation by preexisting human antibodies. In other words, the present specification discloses AAV capsids that arc so- called “epitope masked”, and therefore are not bound by neutralising antibodies that may be present in a subject. In one example, the composition is a pharmaceutical composition comprising one or more of the AAV capsid proteins disclosed herein. In another example, the composition comprises one or more of the AAV capsid proteins disclosed herein and pharmaceutically acceptable carriers and/or buffers.

[0022] Without being bound by theory, it is thought that compositions disclosed herein evade antibody neutralisation due to epitope masking. As used herein, the term “epitope masked” refers to an antigenic sequence of a virus, for example, the AAV capsid (i.e., antigen is bound by antibody) disclosed herein which is engineered to remove or minimize the binding of the antibody. The result is evasion of antibody binding (including, but not limited to, that of neutralising antibodies), without compromising or losing the desired function of the AAV (which can be described and/or quantified as a titre (as a measure of infectivity of a virus) and/or transduction efficiency of the AAV). Maintaining desired function of the virus is important because mutation of the antigen while losing function would not be considered “masking”. A loss of function (either infectivity or reduction in virulence or pathogenicity) would instead be understood as viral attenuation.

[0023 J Such epitope masking can be performed, for example, by inserting one or more mutations within the relevant sections of the viral genome. The person skilled in the art would be able to insert or generate mutations in the viral genome or sections thereof, or within a specific gene, or replace specific nucleotide residues, for example, by inserting extraneous nucleotides (frame-shift mutations) or by replacement/substitution of specific nucleotides. The same mutations can also be generated on the protein level. A person skilled in the art would be aware of how to perform such amino acid substitution on virus particles.

[0024] As used herein, the term “protein” refers to a whole molecule. In contrast, as used herein, the term “peptide” refers to a sub-segment or part of the whole (protein) molecule.

[0025] As used herein, the term “epitope” refers to an immunogenic peptide sequence within a protein (also referred to in this example as an “antigen”). In other words, an epitope refers to a group of amino acids (peptides or proteins) or other chemical groups exposed on the surface of a molecule (the antigen), which can generate an antigenic response and bind an antibody. [0026] The antibody antigenic footprint on different adeno-associated virus (AAV) orthologues were identified using phage display, specifically phage-immunoprecipitation sequencing (PhlP-Seq). An antibody epitope common to all tested AAV serotypes was also identified. AAVs engineered for protein residue substitution in this common epitope were shown to be resistant to neutralisation, if not completely resistant to neutralisation by pre-existing antibodies. The use of this modified adeno-associated virus (AAV) will enable more efficacious gene delivery by AAV through evading undesirable antibody immune response. Thus, in one example, there is disclosed a composition comprising at least one modified adeno- associated viral (AAV) capsid protein, wherein the composition is resistant to neutralisation or is not neutralised by human neutralising antibodies.

[0027] Presented herein is also the development of adeno-associated virus (AAV) capsids that are resistant to neutralization by pre-existing antibodies. The present disclosure describes AAVs as part of compositions that are engineered at a B-cell epitope with amino acid sequences that were previously not known to (1) engage the human antibodies and (2) have been shown to evade humoral immunity when engineered. Additionally, the neutralising antibody resistant AAV variants described herein showed an increase in vitro transduction efficiency in the presence and in the absence of neutralising antibody (N Abs). [0028] The present disclosure describes mutations that are performed to obtain mutated or modified adeno-associated virus (AAV) vectors or virus particles. In one example, the mutations are at residues within epitopes residing in the N-terminal regions of the AAV capsid proteins. These residues of the epitopes referred to herein can be, but are not limited to, the following residues: amino acids 24 to 73, amino acids 48 to 93, amino acids 168 to 217, amino acids 48 to 97, amino-acids 72 to 121, amino acids 120 to 167, and combinations thereof. The numbering disclosed herein is provided in reference to the full- length sequence of the AAV VP1 capsid protein.

[0029] A total of five (5) serum samples from Southeast Asian patients and a commercially pooled serum sample (from 526 healthy donors between the ages 18 to 65) were tested in a phage-based immunoprecipitation assay. After identifying enriched peptides immunoprecipitated by the human antibodies, significant antibody binding sites in the N- and C- terminal parts of different AAV serotypes were detected. The C -terminal part of the AAV capsid containing the VP3 region has previously been described as a hotspot for the antibody binding but the data disclosed herein highlights that all human samples were reactive to epitopes from the N-terminal region of AAV capsids (VP1/ VP2 common regions (from 24 to 217 aa) (Figure 2). Without being bound by theory, it is also thought that mutated phage display tiles show decreased binding to human sera (data not shown).

[0030] The N-terminal was therefore chosen as an epitope because it is highly immunogenic based on the phage display data. In addition, much less is known about the N-terminus since it is unstable and the crystal structure of AAV capsid docs not capture its structure. Also, the data presented herewith has shown that highly efficient immune evasion is obtained by masking this epitope. [0031] Thus, in one example, the at least one modified adeno-associated vit al (AAV) capsid protein is modified at any one or more of residues 24 to 47, 48 to 97, 72 to 121, 120 to 167 and 168 to 217 of an adeno-associated viral (AAV) VP1 capsid protein or an ortholog thereof. As used herein, the term “ortholog” refers to any of two or more homologous gene sequences found in different species related by linear descent,

[0032] In one example, the at least one modified adeno-associated viral (AAV) capsid protein is modified at any one or more of residues selected from the group consisting of positions 24 to 73 and 48 to 93. In another example, the at least one modified adeno-associated viral (AAV) capsid protein is modified at any one or more of residues selected from the group consisting of residues 24 to 93.

[0033] The AAVs disclosed herein can comprise one or more mutations in order to, for example, epitope mask said virus, resulting in modified adeno-associated viral (AAV) capsid proteins. In one example, the at least one modified adeno-associated viral (AAV) capsid protein comprises at least one, at least two, at least three mutations, or more. In another example, the at least one modified adeno-associated viral (AAV) capsid protein comprises at least one mutation. In yet another example, the at least one modified adeno-associated viral (AAV) capsid protein comprises at least two mutations. In a further example, the at least one modified adeno-associated viral (AAV) capsid protein comprises at least three mutations. In one example, the one or more mutations result in the modified adeno-associated viral (AAV) capsid protein being epitope masked. In other words, the one or more mutations result in the modified adeno-associated viral (AAV) capsid protein being able to evade neutralisation by antibodies already present in a host or a subject.

[0034] The results indicated that the mutations did not affect the genome packaging efficiency. Furthermore, it was observed that few mutations resulted in an increase in yield (Figure 3A and Table 2). Various mutations, as well as multiple mutations in the same variant, are contemplated within the scope of the disclosure as presented herewith. Without being bound by theory, mutations can be included in the claimed variant, so long as the mutation(s) do not impact negatively affect the transduction efficiency of the capsid protein disclosed herein.

[0035] As disclosed above, mutations can be inserted on the gene or protein level. In the present disclosure, the mutation is inserted into the viral genome. In one example, the mutation is selected from the group consisting of a “gca” mutation (alanine), a “egg” mutation (arginine), and a “tef ’ mutation (serine). In another example, the mutation is a “gca” mutation (alanine). In another example, the mutation is a “egg” mutation (arginine). In yet another example, the mutation is a “tet” mutation (serine). In yet another example, the mutation is a “gau” or “gac” mutation (aspartic acid). A person skilled in the art would appreciate that due to codon redundancy, other equivalent mutations may be used other than those described in the present disclosure. In one example, the residues arc numbered at positions according to the wild-type sequence. In one example, the wild-type sequence or reference sequence is an adeno-associated viral (AAV) VP1 capsid protein or an ortholog thereof. [0036] In one example, the at least one mutation is present at any one or more of the residues selected from the group consisting of A70, R168, KI 69, R170, L171, N172, F173, G174, Q175, T176, G177, D178, A179, D180, S181, V182, P183, D184, P185, Q186, P187, 1188, G189, E190, P191A, P192, P195, A193, A194, P195, S196, G197, V198, G199, S200, L201 , T202, M203, A204, A205, G206, G207, G208, A209, P210, M211, A212, D213, N214, N215, E216, G217, N215, A70, and combinations thereof.

[0037] In another example, the at least one mutation is selected from the group consisting of A70R, R168A, K169R, R170A, L171 A, N172A, F173A, G174A, Q175A, T176A, G177A, D178A, A179R, D180A, S181A, V182A, P183A, D184A, P185A, Q186A, P187A, I188A, G189A, E190A, P191A, P192A, P195D, A193R, A194R, P195A, S196A, G197A, V198A, G199A, S200A, L201A, T202A, M203A, A204R, A205R, G206A, G207A, G208A, A209R, P210A, M211A, A212R, D213A, N214A, N215A, E216A, G217A, N215S, A70R, and combinations thereof.

[0038] In another example, the at least one “gca” mutation (alanine) is present at any one or more of the residues selected from the group consisting of R168, R170, L171, N172, F173, G174, Q175, T176, G177, D178, D180, S181, V182, P183, D184, P185, Q186, P187, 1188, G189, E190, P191, P192, P195, S196, G197, V198, G199A, S200, L201 , T202, M203, G206, G207, G208, P210, M21 1 , D213, N214, N215, E216, G217, and combinations thereof.

[0039] In another example, the at least one “egg” mutation (arginine) is present at any one or more of the residues selected from the group consisting of A70, K169, A179, A193, A194, A204, A205, A209, A212, A70, and combinations thereof.

[0040] In another example, the at least one “tet” mutation (serine) is present at N215.

[0041] In another example, the at least one “gau” or “gac” mutation (aspartic acid) is present at P195. [0042] In one example, the at least one mutation is D213A. In another example, there are two mutations. In another example, there are two mutations, which are D213A and A70R.

Also disclosed herein are modified adeno-associated viral (AAV) capsid protein comprising the one or more mutations disclosed herein, at the residues disclosed herein. This, in one example, there is described at least one modified adeno-associated viral (AAV) capsid protein is selected from the group consisting of AAV-DJ_R168A, AAV-DJ_K169R, AAV-DJ_R170A, AAV-DJ_L171A, AAV-DJ_N172A, AAV- DJ_F173A, AAV-DJ_G174A, AAV-DJ_Q175 A, AAV-DJ_T176A, AAV-DJ .G177A, AAV-DJ_D178A, AAV-DJ_A179R, AAV-DJ_D180A, AAV-DJJS181A, AAV-DJ_V182A, AAV-DJ_P183A, AAV- DJ D184A, AAV-DJ Pl 85A, AAV-DJ_Q186A, AAV-DJ_P187A, AAV-DJJ188A, AAV-DJ_G189A, AAV-DJ_E190A, AAV-DJ_P191A, AAV-DJ_P192A, AAV-DJ_P195D, AAV-DJ_A193R, AAV- DJ A194R, AAV-DJ P195A, AAV-DJ_S196A, AAV-DJ_G197A. AAV-DJ_V198A, AAV-DJ_G199A, AAV-DJ_S200A, AAV-D.T L201 A, AAV-DJ_T202A, AAV-DJ_M203A, AAV-DJ_A204R, AAV- DJ_A205R, AAV-DJ _G206A, AAV-DJ _G207A, AAV-DJ _G208A, AAV-DJ_A209R, AAV-DJ_P210A, AAV-DJ_M211 A, AAV-DJ A212R, AAV-DJ_D213A, AAV-DJ_N214A, AAV-DJ_N215A, AAV- DJ_E216A, AAV-DJ_G217A, AAV-DJ_N215S, AAV2_D213A, AAV9_D213A, AAV-DJ_A70R, and AAV-DJ_A70R_D213A. Non-exhaustive examples of such adeno-associated viral (AAV) capsid proteins can be found in Table 1.

[0043] Table 1: AAV-DJ variants

[0044] In one example, the one adeno-associated viral (AAV) capsid protein disclosed herein comprises or is selected from the group consisting of SEQ ID Nos 30 to 57 and 61 to 62, or is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID Nos 1 to 29 and 58 to 60. [0045] In one example, the at least one adeno-associated viral (AAV) capsid protein does not comprise or consist of a B-cell epitope. In another example, the modified adeno-associated viral (AAV) capsid proteins disclosed herein comprise or are selected from the group consisting of the sequences disclosed herein. See, for example, the sequence listing section of this application.

[0046] The in vitro transduction efficiencies of the variants in HEK293, HepG2, and SK-Hep-1 cell lines were tested. The findings indicated that the mutations, with the exception of R170A, did not affect the transduction efficiency of the AAV-DJ variant.

[0047] Few mutants displayed an improvement in transduction efficiency in HEK293T, and SK-Hep1 cells when compared to the wild-type AAV-DJ vector. (Table 2) It is also noted that transduction efficacy may vary in terms of distribution of variants across different tissues and cell populations, as well as in factors like, but not limited to, vector yield, transduction efficiency in vitro and biodistribution in vivo.

[0048] The ability of the single AAV variants to avoid neutralization by human sera (pooled from 526 healthy donors between the ages 18 to 65) was evaluated using an in vitro neutralisation assay. Stringent conditions were used by including undiluted serum (1:1) and dilutions up to 1:316. The exclusion criterion used in ongoing clinical trials for indications requiring systemic administration of AAV is a NAb titre of 1: 5.

A number of variants (R168A, T176A, P183A, P187A, P192A, P195A, S196A, G197A, L201A, A205R, N214A, E216A, G217A) showed improved neutralization resistance than WT AAV-DJ (one to two log decrease in NAb titre). In particular, the D213A variant displayed a complete resistance to neutralisation by pre-existing antibodies (titre 1:3.16) (Figure 4B), which with the current exclusion criteria, the patients will be considered as compatible subjects (similar to naive / no pre-existing antibodies against the AAV). Hence, the compositions and modified AAV capsid proteins disclosed here exhibit evasion of human neutralising antibodies.

[0049] Table 2: Summary of vector titre yield, in vitro transduction efficiency and neutralising antibody (Nab) titre.

[0050] Table 3: Non-Alanine substitutions for avoiding neutralising antibody (Nab) recognition.

[0051] Next, it was tested to see whether the D213A substitution enables escape from pre-existing NAbs in human serum across different AAV serotypes. First, AAV2_D213A and AAV9_D213A were produced and the genome titres determined. Similar to previous results obtained with AAV-DJ_D213A, the D213A mutation did not negatively impact bioproduction yield. Notably, the substitution D213A resulted in a reduction in neutralization by pre-existing antibodies, as evidenced by a drop of two logs in Nab titre for AAV2 D213A (titre 1:10 versus 1:316 for WT AAV2) and one log for AAV9_D213A (1:1 versus 1:3.16 for WT AAV9, Table 3).

[0052] Building on the results of epitope-mapping disclosed herein, residues 24-73 and 48-93 were selected for further analysis. Specific residues were identified and selected based on their conservation across a range of AAV serotypes. Following this, AAV-DJ variants were produced and evaluated in their in vitro transduction efficiency. A number variants exhibited infectious capabilities. Subsequently, their potential to evade neutralization by pre-existing NAbs in human sera was assessed using an in vitro assay. Notably, the AAV-DJ_A70R variant demonstrated resistance to pre-existing NAbs, with a one-log reduction in Nab titre (1:10) compared to wild-type AAV-DJ (Table 3).

[0053] It was sought to determine if combining the A70R and D213A mutations was capable of synergistically enhancing evasion from pre-existing NAbs in human serum. To this end, the double mutant was prodeed. It was observed that the A70R_D213A mutation did not impact either the bioproduction yield (Table 3) or the in vitro transduction efficiency. The results were similar to those observed for both AAV- DJ_D213A and AAV-DJ_A70R_D213A, with the Nab titre further dropping to 1:3.16 compared to WT AAV-DJ (1:31.6 - 1:100, Table 4, Figure 6).

[0054] Table 4: Additional variants for avoiding Nab recognition.

[0055] Importantly, maintaining desired function of the virus is important because mutation of the antigen while losing function would not be considered “masking”. Thus, in one example, the at least one mutations present in the modified adeno-associated viral (AAV) capsid protein do not negatively impact the transduction efficiency of modified AAV capsid protein. Tn other words, the modified adeno-associated viral (AAV) capsid proteins disclosed herein arc capable of infection host cells, as well as transfection their payload into said host cell, thereby resulting in expression of said payload by the host cell.

[0056] In another example, the present disclosure describes a nucleic acid sequence encoding the composition disclosed herein, or encoding the one or more modified adeno-associated viral (AAV) capsid proteins as described herein.

[0057] Further disclosed herein is, in one example, a vector comprising one or more nucleic acid sequences disclosed herein. In another example, there is disclosed a vector encoding the one or more modified adeno-associated viral (AAV) capsid proteins disclosed herein.

[0058] Described herein is, for example, a host cell comprising the vector disclosed herein or the nucleic acid sequence disclosed herein. In another example, there is disclosed an adeno-associated virus particle comprising the modified AAV capsid proteins disclosed herein.

[0059] Another use described in the present disclosure is the use of the compositions, vectors, or modified AAV capsid proteins, adeno-associated virus particles disclosed herein in situations where binding to any antibodies present is not desirable. For example, in gene therapy, the vector (for example, a payload-carrying virus particle) needs to reach its intended target (the target cell), among others, in order to be effective. For this reason, vectors that are bound to by antibodies are not considered to be effective in their intended application. Thus, also disclosed herein is the composition, the nucleic acid, the vector, the host cell, or the adeno-associated virus particle disclosed herein, for use in gene therapy. In another example, there is disclosed a method of treating a subject in need thereof, the method of treating a subject comprising administering to the subject a therapeutically effective amount of the composition the nucleic acid, the vector, the host cell, or the adeno-associated virus particle disclosed herein. In yet another example, there is disclose use of the composition, the nucleic acid, the vector, the host cell, or the adeno-associated virus particle disclosed herein, in the manufacture of a medicament, wherein the medicament is used in or for gene therapy. In a further example, the present disclosure describes use of the composition, the nucleic acid, the vector, the host cell, or the adeno-associated virus particle disclosed herein, in the manufacture of a medicament for gene therapy.

[0060] A further use described herein is when an AAV is to be administered to a subject more than once. Upon first administration, the subject may develop antibodies to the AAV that had been administered, thereby precluding the same subject from receiving further doses or administrations of the same AAV in future. Thus, in one example, the modified AAV capsid proteins disclosed herein do not elicit an immune response in the host. In another example, there is also described a method of preventing an immune response to an engineered adeno-associated virus, the method comprising administering to a subject an engineered adeno-associated virus comprising at least one modified adeno-associated viral (AAV) capsid protein.

[0061] Also disclosed herein is a method of identifying an adeno-associated viral (AAV) capsid protein or peptide which is resistant to neutralisation or is not neutralised by human neutralising antibodies, the method comprising (a) contacting the adeno-associated viral capsid protein or peptide with sera known to contain adeno-associated virus neutralising antibodies, thereby allowing the neutralising antibodies to bind to an epitope comprised within the adeno-associated viral capsid protein or peptide; (b) removing the neutralising antibody-bound epitope from the adeno-associated viral capsid protein or peptide; and (c) identifying the adeno-associated viral capsid protein or peptide that did not bind to the neutralising antibodies. In another example, the adeno-associated viral (AAV) capsid protein or peptide is selected from the group consisting of AAV-DJ, AAV1, AAV2, AAV3, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 AAV13, or an ortholog thereof.

[0062] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0063] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers to, including mixtures and combinations thereof.

[0064] As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

[0065] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not he construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0066] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0067] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0068] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION

Identification of epitopes on AA Vs with human sera

[0069] The pre-existing anti-AAV antibodies pose a serious challenge for the efficacy of gene transfer mediated by AAV vectors. While there have been a few studies characterizing the extent of pre-existing immunity in human populations, the identification of the exact epitopes involved has not yet been comprehensively evaluated. To characterize the anti-AAV antibody repertoire in human subjects, a phage display library composed of approximatively 10,000 unique tiles across different AAV capsid orthologues was generated (Figure 1). This library also contains tiles from known Cas9, Casl2 and Casl3 proteins. A python script described by Mohan et al. Nature protocols 2018 (PMTD: 30190553) was used. It is noted that the phage display tiles referred to herein also consist of segments from the AAV orthologues. The phage display can comprise sequences that differ from the naturally occurring ones, such as single residue or multiple residues substitutions/insertions/deletions. This library has the additional advantage of probing antibody binding to sequence variants. Design, Production and Characterization of the phage library

[0070] The phage library contained the protein sequences from the most common AAV serotypes (AAV1- AAV13, AAV DJ), together with the most common orthologues of Cas9, Casl2 and Casl3. Each protein was computationally divided into 49-amino acid peptide tiles with a 24-amino acid overlapping window. The amino acid sequences of these tiles were then reverse translated with an E. coli codon optimized algorithm called “pepsin” into DNA sequences. This library was provided as a Twist library, with the addition of universal forward, reverse primers, and with EcoRl and Hindlll digestion sites inserted to the fragments. The oligo pool was amplified with Q5 Polymerase. The library was then digested with EcoRl and Hindlll for 1 hour at 37 °C and ligated into pre-digested T7Sclcct 10-3b Vector Arms (EMD; 16 hours at 16 °C), before being packaged into phages with T7Select® Packaging Extract (EMD). The phage library titre was determined by the plaque assay and the library was further amplified using BLT5403 E. coli cultures. The library was then amplified and concentrated by precipitation with 20% PEG8000 and 2M NaCl, before being resuspended in storage buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 6 mM MgClz).

[0071] The library was tested with five (5) different, individual patient sera and a commercially available pooled serum sample that had been collected from 526 patients between the ages of 18 to 65.

[0072] For each sample subjected to the PhlP-seq assay, 10" pfu of phage library was mixed with sera containing 2 pg of IgG (quantified by ELISA). The samples were incubated for 18 hours at 4°C. As a positive control, 2 pg of commercially available SpCas9 polyclonal antibodies was used. Two technical replicates were performed for each sample. Next, 20pl of protein A and 20pl of protein G Dynabeads were added to each tube and incubated for another 4 hours at 4°C. Beads were washed twice with 170 pl of IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40). After removing the wash solution, the beads were directly used for polymerase chain reaction (PCR). For Illumina library preparation, phage DNA was PCR-amplified with Q5 High-Fidelity DNA polymerase (New England Biolabs) in two rounds to produce Illumina libraries containing adaptor sequences and barcodes for multiplexing. The first round of polymerase chain reactions (PCRs) was performed using immunoprecipitated beads as template and with the primers amplifying the tiles.

[0073] In the second polymerase chain reaction (PCR), 2 pL of first reaction was used as template, and unique dual-indexed primers (NEBNext® Multiplex Oligos for Illumina) were used for barcoding. The PCR reactions were purified using SPRI Beads. The quality of the DNA fragments was confirmed on Bioanalyzer and the library was quantified by Qubit. The samples were then pooled in equimolar quantities and submitted for sequencing on a HiSeq with paired-end reads. To determine the significance of enriched peptides from noise that may be introduced by non-specific binding, the sample sets were fit to the Gamma Poisson model in the phip-stat Python package provided by the Laserson Lab (https://github.com/lascrsonlab/phip-stat). In brief, after sequencing, the reads were mapped to the original library using Bowtie2. SAM tools were used to count the number of reads for each peptide in the input and for each sample. The gamma Poisson model was fitted to the distribution of the output samples. This mode was used to generate (-logio) P values for the probability of enrichment of each peptide. For each run, data from mock-IP controls (no antibody) were included with the patient sample data to better account for the abundance and non-specific binding associated with each peptide in the phage library.

[0074] In addition, the generalisable method to mask the other identified epitopes consists of (1) identifying the epitopes via phage display, (2) optionally selecting the strongest / most immune dominant epitopes for masking (as performed in the example described herein), (3) modifying the amino acid sequences of selected epitopes to eliminate the antibody-recognised sequences, (4) validating immune evasion and retention of transduction function.

AAV capsids engineered for immune evasion by epitope-masking

[0075] Based on the epitope-mapping, residues 168 to 217 of the AAV VP1 capsid protein were selected for epitope-masking. These residues had not been previously described to influence the biological properties of AAV vector, nor had these residues been previously known as an antibody epitope of any AAV serotypes. Multiple, evolved AAV variants were generated using alanine screening mutagenesis (Table 1 ) using the pRep2-AAVCapDJ as a template. The PCR product was then treated with Dpnl and T4 polynucleotide kinase at 37 °C for 1 hour before being ligated using T4 DNA ligase and incubated overnight at 16°C. Ligated product was then transformed into chemically competent 5-alpha E. coli. The generated plasmid was Sanger sequenced for sequence verification and the correct clone was amplified and purified using plasmid plus maxi kit. Using this method, the AAV-DJ variants shown in Table 1 were generated.

[0076] Table 5: Primer sequences for AAV-DJ plasmids cloning.

[0077] The AAV-DJ and the different variants were produced containing a self-complementary GFP genome driven by a ubiquitous Casi promoter. The genome titre of AAV-DJ variants and the wild-type (WT) AAV-DJ vectors were evaluated using quantitative PCR (qPCR).

AAV vector production

[0078] AAV vectors were produced by triple transfection of HEK293T cells and purified via an iodixanol gradient. Cells were transfected with 20 pg of a helper plasmid, 10 pg of an ITR cargo plasmid, and 10 pg of rep-cap plasmid. 24 hours later, the medium was changed to a fresh culture medium containing 2% foetal bovine serum (FBS). Three days after transfection, cells were collected and resuspended in lysis buffer (Tris HC1 pH 7.5, 2 mM MgCh, 150 mM NaCl) and lysed in three freeze/thaw cycles. The supernatant was collected and treated with 50 U/ml of benzonase and 1 U/ml of RNase cocktail for 30 minutes at 37 °C to remove unpackagcd nucleic acids. After incubation, the lysate was loaded on top of a discontinuous density gradient (15%, 25%, 40%, and 60%) and ultracentrifuged at 54,000 rpm, at 18 °C, for 1.5 hours, on a Type 70 Ti rotor. The 40% fraction was extracted and dialyzed with IxPBS (pH 7.2) with 0.001% pluronic acid, using Amicon ultra-15 (100 kDa MWCO). The vectors were titrated against an AAV2 RSM vector by quantitative PCR using primers and probe binding within the AAV2 ITR.

In vitro neutralisation assay

[0079] HEK293 expressing adenoviral E4 protein in a doxycycline-inducible manner were seeded in 96-well plate at a density of 2xl0⁴ cells/well in 100 pL DMEM containing 10% FBS. The next day, the vectors prepared at a multiplicity of infection (MOI) of 20000 and were pre -mixed with a serial dilution of human serum and incubated for 1 hour at 37°C. As a positive control, the vector was diluted with DMEM. 10 pL of the mixture was added to the wells and incubated for 48 hours at 37 °C. The total GFP protein was quantified using a GFP quantification kit (Biovision) on a multi-well plate reader (Tecan). The neutralising titre was determined as the highest sample dilution at which at least 50% inhibition occurred. SEQUENCE LISTING

[0080] The following sequences are disclosed in the present application.

Claims

1. A composition comprising at least one modified adeno-associated viral (AAV) capsid protein, wherein the composition is resistant to neutralisation or is not neutralised by human neutralising antibodies, wherein the at least one modified adeno-associated viral (AAV) capsid protein is modified at any one or more of residues 24 to 47, 48 to 97, 72 to 121, 120 to 167 and 168 to 217 of an adeno-associated viral (AAV) VP1 capsid protein or an ortholog thereof.

2. The composition of claim 1 , wherein the at least one modified adeno-associated viral (AAV) capsid protein is modified at any one or more of residues selected from the group consisting of residues 24 to 93.

3. The composition of claim 2, wherein the at least one modified adeno-associated viral (AAV) capsid protein is modified at any one or more of residues selected from the group consisting of residues 24 to 73 and 48 to 93.

4. The composition of any one of the preceding claims, wherein the at least one modified adeno- associated viral (AAV) capsid protein comprises at least one, at least two, at least 3 mutations or more.

5. The composition of any one of the preceding claims, wherein the mutation results in the modified adeno-associated viral (AAV) capsid protein being epitope masked.

6. The composition of any of the preceding claims, wherein the mutation is selected from the group consisting of a “gca” mutation (alanine), a “egg” mutation (arginine), a “tet” mutation (serine), and a “gau” or “gac” mutation (aspartic acid).

7. The composition of claim 6, wherein the at least one mutation is present at any one or more of the residues selected from the group consisting of A70, R168, K169, R170, L171, N172, F173, G174, Q175, T176, G177, D178, A179, D180, S181, V182, P183, D184, P185, Q186, P187, 1188, G189, E190, P191A, P192, P195, A193, A194, P195, S196, G197, V198, G199, S200, L201, T202, M203, A204, A205, G206, G207, G208, A209, P210, M211, A212, D213, N214, N215, E216, G217, N215, A70, and combinations thereof.

8. The composition of any one of claims 6 to 7, wherein the at least one mutation is selected from the group consisting of A70R, R168A, K169R, R170A, L171A, N172A, F173A, G174A, Q175A, T176A, G177A, D178A, A179R, D180A, S181A, V182A, P183A, D184A, P185A, Q186A, P187A, Il 88A, G189A, E190A, P191A, P192A, P195D, A193R, A194R, P195A, S196A, G197A, V198A, G199A, S200A, L201A, T202A. M203A, A204R, A205R, AAV-DJ_G206A, G207A, G208A, A209R, P210A, M211A, A212R, D213A, N214A, N215A, E216A, G217A, N215S, A70R, and combinations thereof.

9. The composition of any one of claims 6 to 8, wherein the at least one “gca” mutation is present at any one or more of the residues selected from the group consisting of R168, R170, L171 A, N172, F173, G174, Q175, T176, G177, D178, D180, S181, V182, P183, D184, P185, Q186A, P187, 1188, G189, E190, P191, P192, P195, S196, G197, V198, G199, S200, L201, T202, M203, G206, G207, G208, P210, M211, D213A, N214, N215, E216, G217, and combinations thereof.

10. The composition of any of the preceding claims, wherein the at least one modified adeno-associated viral (AAV) capsid protein is selected from the group consisting of AAV-DJ_R168A, AAV- DJ K169R, AAV-DJ_R170A, AAV-DJ_L171 A, A AV-DJ_N172A, A AV-DJ_F173 A, AAV- DJ_G174A, AAV-DJ_Q175A, AAV-DJ_T176A, AAV-DJ_G177A, AAV-DJ_D178A, AAV- DJ A179R, AAV-DJ D180A, AAV-DJ_S181A, AAV-DJ_V182A, AAV-DJ_P183A, AAV- DJ_D184A, AAV-DJ_P185A, AAV-DJ_Q186A, AAV-DJ_P187A, AAV-DJJ188A, AAV- DJ G189A, AAV-DJ E190A, AAV-DJ_P191A, AAV-DJ_P192A, AAV-DJ_P195D, AAV- DJ_A193R, AAV-DJ_A194R, AAV-DJ_P195A, AAV-DJ_S196A, AAV-DJ_G197A, AAV- DJ V198A, AAV-DJ G199A, AAV-DJ_S200A, AAV-DJ_L201A, AAV-DJ_T202A, AAV- DJ_M203A, AAV-DJ_A204R, AAV-DJ_A205R, AAV-DJ_G206A, AAV-DJ_G207A, AAV- DJ G208A, AAV-DJ_A209R, AAV-DJ_P210A, AAV-DJ_M211A. AAV-DJ_A212R, AAV- DJ D213A, AAV-DJ_N214A, AAV-DJ_N215A, AAV-DJ_E216A, AAV-DJ_G217A, AAV- DJ_N215S, AAV2_D213A, AAV9_D213A, AAV-DJ_A70R, and AAV-DJ_A70R_D213A.

11. The composition of any one of the preceding claims, wherein the at least one adeno-associated viral (AAV) capsid protein comprises or is selected from the group consisting of SEQ ID Nos 30 to 57 and 61 to 62, or is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID Nos 1 to 29 and 58 to 60.

12. A nucleic acid sequence encoding the composition of any one of claims 1 to 11, or encoding the one or more modified adeno-associated viral (AAV) capsid proteins according to any one of claims 1 to 11.

13. A vector comprising the nucleic acid sequence of claim 12.

14. A host cell comprising the vector of claim 13 or the nucleic acid sequence of claim 12.

15. An adeno-associated virus particle comprising the AAV capsid proteins according to any one of claims 1 to 11.

16. The composition according to any one of claims 1 to 11, the nucleic acid of claim 12, the vector of claim 13, the host cell of claim 14, or the adeno-associated virus particle of claim 15, for use in gene therapy.

17. A method of identifying an adeno-associated viral (AAV) capsid protein or peptide which is resistant to neutralisation or is not neutralised by human neutralising antibodies, the method comprising a. contacting the adeno-associated viral capsid protein or peptide with sera known to contain adeno-associated virus neutralising antibodies, thereby allowing the neutralising antibodies to bind to an epitope comprised within the adeno-associated viral capsid protein or peptide; b. removing the neutralising antibody-bound epitope from the adcno-associatcd viral capsid protein or peptide; and c. identifying the adeno-associated viral capsid protein or peptide that did not bind to the neutralising antibodies.

18. The method of claim 17, wherein the adeno-associated viral (AAV) capsid protein or peptide is selected from the group consisting of AAV-DJ, AAV1, AAV2, AAV3, AAV3, AAV4, AAV5, AAV6. AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 AAV13, or an ortholog thereof.

19. A method of preventing an immune response to an engineered adcno-associatcd virus, the method comprising administering to a subject an engineered adeno-associated virus comprising at least one modified adeno-associated viral (AAV) capsid protein according to claims 1 to 11.