Modular platform for gene therapy

Abstract

The present invention relates to the field of modular platforms, such as viral vectors. In particular, the present invention relates to a recombinant adeno- associated virus (AAV) capsid or particle comprising an engineered virion protein (VP), wherein the engineered VP comprises at least one cell-type specific binding moiety located in variable region 8 (VR8). The invention also relates to a recombinant AAV particle for use as a medicament.

Description

[0001] 84935PC01

[0002] 1

[0003] Modular Platform for Gene Therapy

[0004] Technical field of the invention

[0005] The present invention relates to the field of modular platforms, such as viral vectors. In particular, the present invention relates to a recombinant adeno- associated virus (AAV) capsid or particle comprising an engineered virion protein (VP), wherein the engineered VP comprises at least one cell-type specific binding moiety located in variable region 8 (VR8). The invention also relates to a recombinant AAV particle for use as a medicament.

[0006] Background of the invention

[0007] Adeno-associated viruses (AAVs) are used as delivery vehicles in gene therapies with increasing clinical success. By replacing the native gene of the AAV with a therapeutic gene, genetic diseases can be treated by having the AAV infect a malfunctioning tissue.

[0008] Despite this recent success, challenges with AAV gene therapies remain. An issue with current AAV gene therapies is that the naturally occurring AAV serotypes, although used in approved AAV drugs today, may not be selective enough for certain indications or desired tissues of therapeutic interest.

[0009] Common for almost all natural AAV serotypes is their ability to transduce a wide array of cells and tissues. This poses a safety risk, especially where a selective approach is required such as in targeted cancer treatment. The endogenous lack of tissue specificity also leads to increased dosage regimes to achieve a therapeutic effect in the desired tissue. High doses of AAVs increase the risk of severe side effects for patients as well as making AAVs some of the world's most expensive therapies.

[0010] WO 23 / 214346 Al discloses adeno-associated virus (AAV) VP2 fusion polypeptides comprising an AAV VP2 capsid polypeptide and a polypeptide ligand, wherein the ligand can be an affibody molecule, which is fused to the N-terminus of the AAV VP2 capsid. However, the challenge of attaching a ligand to the N-terminus of VP2 is serotype-dependent in terms of functionality of the ligand, thus restricting the functional attachment of ligands, such as affibody molecules, to a limiting variety of AAV serotypes. 84935PC01

[0011] 2

[0012] Hence, an improved recombinant AAV particle by altering the AAV tropism to make the AAVs cell-type specific could lead to more effective gene therapies, reducing doses, potential side-effects, as well as opening up for new areas of application would be advantageous, and in particular a modular cell-type specific recombinant AAV particle would be advantageous.

[0013] Summary of the invention

[0014] The attachment of at least one cell-type specific binding moiety to a variable region, such as VR8, of the AAV capsid protein provides a feasibility to integrate cell-type specific binding moieties across a broad variation of AAV serotypes. Unmodified AAVs generally have a very broad tropism, which leads to increased dosage regimes to reach clinical effect, which in turn increases the risk of patient side effects as well as drives up drug costs. The improved selectivity of the recombinant AAV capsid or particle according to the present invention allows specific targeting of cells and thereby leads to more effective therapies, such as gene therapies, reducing administered doses, and reducing potential side-effects. Particularly, the recombinant AAV of the present invention is a modular platform for retargeting of recombinant AAV particles towards specific cell surface molecules, such as receptors, and thereby obtaining an improved recombinant AAV particle, which reduces off-target effects and improves on-target selectivity. The recombinant AAV capsid or particle of the present invention demonstrates potential for use as a medicament and for use in the treatment of diseases and conditions, such cancer and genetic diseases.

[0015] In particular, the present invention relates to recombinant AAV capsid or particles, which have been genetically modified to express cell-type specific affibody molecules, i.e., the affibody-modified AAV particles can target any cell or tissue of choice depending on the affibody used. The invention also relates to a system for using said recombinant AAV particles to deliver nucleic acids as gene therapy to target cells or tissue. The affibody-modified AAV particles tackles the issues presented above by making the recombinant AAV particles selective for certain cells, and thereby lowering the number of AAV particles required to infect a certain number of cells and reducing off-target effects. 84935PC01

[0016] 3

[0017] Importantly, the cell-type specific binding moiety having a protein structure of a three-helix bundle, allows for the system to be modular, since the same positions in the three-helix bundle can be mutated to target different cell surface receptors. Additionally, since the Affibody molecules could be integrated without any modifications to the VR8 or the Affibody sequence, and since there is a vast number of published Affibody molecules targeting an almost equally vast number of cell receptors, the modularity of the platform is further proven. The present invention exemplifies the modularity of the recombinant AAV platform by exchanging functional affibody molecules for targeting different surface proteins. It is surprising that such a structure of a three-helix bundle could be accommodated at / in a VR8 site, given that the N- and C-terminal ends of a three- helix bundle are oriented approximately 180° apart - posing a structural challenge for integration into a loop region (VR8).

[0018] Thus, an object of the present invention relates to the provision of an improved modular platform for targeting recombinant AAV particles towards specific cell surface molecules, thereby reducing off-target effects and improving on-target selectivity.

[0019] In particular, it is an object of the present invention to provide a recombinant AAV capsid or particle that solves the above-mentioned problems of the prior art with improved selectivity, which allows specific targeting of cells and thereby leading to more effective therapies, such as gene therapies, reducing administered doses, and reducing potential side-effects.

[0020] Thus, one aspect of the invention relates to a recombinant adeno-associated virus (AAV) capsid comprising an engineered virion protein (VP), wherein the engineered VP comprises at least one cell-type specific binding moiety located in variable region 8 (VR8), wherein the cell-type specific binding moiety comprises a three-helix bundle and a length selected from the range of 40 to 150 amino acids..

[0021] Another aspect of the present invention relates to a recombinant adeno- associated virus (AAV) particle comprising the recombinant AAV capsid according to the present invention. 84935PC01

[0022] 4

[0023] Yet another aspect of the present invention is to provide a system for delivering at least one nucleic acid to a cell determined by a cell-type specific binding moiety, said system comprises a recombinant adeno-associated virus (AAV) particle according to the invention.

[0024] Still another aspect of the present invention is to provide the recombinant AAV particle according to the present invention or the system according to the present invention for use as a medicament.

[0025] A further aspect of the invention relates to the recombinant AAV particle according to the present invention or the system according to the present invention for use in the treatment, alleviation, and / or prevention of a disease selected from the group consisting of cancer, Alzheimer's disease, Parkinson's disease, epilepsy, Autosomal dominant polycystic kidney disease (ADPKD), Nephronophthisis (NPHP), lipoprotein lipase deficiency, Leber's congenital amaurosis, Spinal muscle atrophy (SMA), Haemophilia A, Haemophilia B, Duchenne muscular dystrophy (DMD), and Aromatic L-amino acid decarboxylase (AADC) deficiency, preferably cancer.

[0026] Yet another aspect of the invention relates to a method for producing the recombinant AAV particle according to the present invention, said method comprising the steps: al) providing a first genetic construct comprising a nucleotide sequence encoding an engineered virion protein (VP) comprising a cell-type specific binding moiety located in variable region 8 (VR8), or a2) providing a first genetic construct encoding an adeno-associated virus (AAV), wherein the start codon of a virion protein (VP) is mutated, and providing a second genetic construct comprising a nucleotide sequence encoding an engineered VP comprising at least one cell-type specific binding moiety located in variable region 8 (VR8), b) transfecting the first genetic construct of step al) or the first genetic construct and the second genetic construct of step a2) into host cells 84935PC01

[0027] 5 in conditions suitable for said host cells to produce AAV particles, and c) isolating AAV particles, wherein the mutated VP and the engineered VP are the same VP variant.

[0028] Brief description of the figures

[0029] Figure 1

[0030] Figure 1 shows (A) a schematic illustration of the CAP genes used for VP- expression of Z-AAVs and controls. dHSPG indicates mutations R585 / 588A in AAV2 variants. The affibody molecules are attached only to VP2, the Z-VP2 construct is expressed separately from the AAV capsid gene, and (B) a schematic illustration of bispecific VP2 (BiZ-VP2). The affibody molecules (Z) were attached to both VR4 and VR8 on VP2 of dHSPG AAV2. For example, the VR4 affibody molecule is ZHER2, and the VR8 affibody molecule is ZHERS.

[0031] Figure 2

[0032] Figure 2 shows number of purified capsids and viral genomes of anti-AAVX- purified (A) AAV2 and (B) AAV8 samples. Titres are presented as the number of viruses purified per 10 cm culture plate used in production. (C) Western blots of the purified AAV preparations with annotated VP bands. D) Average VP-ratio of purified AAV2 and AAV2-derived AAV preparations quantified by image analysis of annotated bands' intensities in Figure 2C. E) Average VP-ratio of purified AAV8 and AAV8-derived AAV preparations quantified by image analysis of annotated bands' intensities in Figure 2C.

[0033] Figure 3

[0034] Figure 3 shows Random sampling of Wasserstein distances for (A) AAV2 and (B) AAV8 samples respectively. Axis shows relative Wasserstein distances between samples.

[0035] Figure 4

[0036] Figure 4 shows (A) Target-ELISA of HER2-specific Z-AAV2 and Z-AAV8 with normalised HER2-binding signal (grey) to anti-AAVX capsid signal (black). Error bars show one standard deviation, average of triplicates for AAV2-variants, duplicates for AAV8-variants. (B) Percent transduced cells in surface display on ExpiCHO-cells displaying no construct (WT), the extracellular domain of HER2 on a 84935PC01

[0037] 6

[0038] GPI anchor (HER2-GPI) or a control domain (RBD-GPI). Transfection efficiencies of the GPI-constructs at the time of AAV addition displayed below the graph. (C) Transduction assay on cancer cell lines (NCI-N87, SK-BR-3, MCF-7) as well as a control fibroblast cell line (hDFn). The gradient indicates relative infectivity from least (black) to the most infectious variant (white) and the displayed number denotes percentage of cells GFP-positive. Average of duplicates. Number of viral genomes per cell indicated on the left. (D) Multiplex transduction assay on surface display ExpiCHO-cells using VR8 Z-AAV2 variants. X-axis denotes target proteins attached to cell surface by GPI-anchoring. Transfection efficiencies of the GPI- constructs at the time of AAV addition displayed below the graph. (E) Transduction assay on cancer cell lines using VR8 Z-AAV2 variants and cell lines NCI-N87, SK-BR-3, MCF-7 and hDFn (control). Colour gradient indicates relative infectivity from least (white) to the most infectious variant (black) and the displayed number denotes percentage of cells GFP-positive. Average of duplicates. Number of viral genomes per cell indicated on the left.

[0039] Figure 5

[0040] Figure 5 shows normalised alamarBlue signal of (A) NCI-N87, (B) SK-BR-3 and (C) hDFn to "Cells only" (cells without AAV addition) for no GCV addition (dark grey circles and bars), 10 pM GCV (stripes squares and bars) and 100 pM GCV (light grey triangles and bars). Mean of six replicates shown, error bars represent one standard deviation. Statistical analysis by two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

[0041] Figure 6

[0042] Figure 6 shows target-binding of BiZ-AAV variants as well as controls with normalised HER2-(stripes), HER3-(light grey) and IGFlR-(dots) binding signal to anti-AAVX capsid signal (black). (A) shows BiZ-AAVs and bivalent controls, (B) shows single Z-AAV variants against the same targets. Error bars show one standard deviation. Values are given as mean of duplicates in (A) and mean of triplicates in (B). (C) Transduction assay of MCF-7 cells with BiZ-AAVs and controls. The mean of duplicates is shown, and percentage transduced cells is displayed for each AAV variant. Colour gradient indicates lowest (black) to highest (white) transduction. 84935PC01

[0043] 7

[0044] Figure 7

[0045] Figure 7 shows (A) schematic illustration of the affibody molecule (Z) construct grafts in VP2 and their different linker (L) lengths, (B) Target-ELISA of HER2- specific Z-AAV8s with varying linker lengths between the AAV and the affibody molecule (Z). HER2-binding signal (stripes) is normalised to anti-AAVX capsid signal (black). Error bars show one standard deviation, average of duplicates, (C) Percent transduced cells in surface display on ExpiCHO-cells displaying no construct (WT), the extracellular domain of HER.2 on a GPI anchor (HER2-GPI) or a control domain (RBD-GPI). Transfection efficiencies of the GPI-constructs 48 h post transfection displayed below the graph, (D) Number of purified capsids and viral genomes of anti-AAVX-purified AAV samples. Titres are presented as the number of viruses purified from three 10 cm culture plates used in production, except for 3L-Z-1L where only two 10 cm culture plates were harvested.

[0046] Figure 8

[0047] Figure 8 shows (A) schematic illustration of linker-free (noL) fusion of two affibody molecules to VP2 in the BiZ-AAVs, (B, C) Target-binding of normalised HER2- (dots), HER3- (squares) and IGF1R- (stripes) binding signal to anti-AAVX capsid signal (black). Monovalent Z-AAVs without linkers as well as Z-AAVs with linkers (B), and BiZ-AAVs with or without linkers (C). Error bars show one standard deviation, bars mean of technical duplicates, (D) Multiplex transduction assay of surface display ExpiCHO-cells using HER2-binding and / or HER3-binding Z-AAV2 and BiZ-AAV2 variants with or without linkers. Transfection efficiencies of the GPI-constructs at the time of AAV addition displayed below the graph, (E) Transduction assay of MCF-7 with BiZ-AAVs and controls. The mean of technical duplicates is shown and percentage transduced cells is displayed for each AAV variant. Colour gradient indicates transduction from lowest (white) to highest (black) and the displayed number denotes percentage of cells GFP-positive. The asterisks indicate the most infectious variant.

[0048] Figure 9

[0049] Figure 9 shows (A) description of in vivo experiment, (B) Average weight differences after injection in percent compared to Day 0 for AAV2 (dots), VR8 noL ZIGFIR -AAV2 (squares), VR8 noL ZpDGFRb-AAV2 (upward triangle) or PBS control (downward triangle). Dots show average of six biological replicates; error bars show one standard deviation, (C-D) eGFP concentration (pg / ml) in the tissue 84935PC01

[0050] 8 homogenates from liver, brain (left hemisphere), pancreas, kidney, heart, muscle (left hindlimb), spleen and lung from six biological replicates. Error bars show one standard deviation. Statistical analysis by one-way ANOVA, followed by Tukey's multiple comparisons test with a single pooled variance. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, (E, F) Liver, lung and muscle from (C) separated by gender (E) or above / below average weight of 28g at time of injection (F). Statistical analysis by two-way ANOVA, followed by Sidak's multiple comparisons test with a single pooled variance. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0051] The present invention will now be described in more detail in the following.

[0052] Detailed description of the invention

[0053] Definitions

[0054] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0055] Cell-type specific binding moiety

[0056] As used herein, a "cell-type specific binding moiety" is a molecule or molecular structure that can selectively bind to a particular type of cell. The specificity is achieved by targeting markers or receptors present on the surface of the target cells.

[0057] It is to be understood that the cell-type specific binding moiety located in VR8 means that, on protein level, the cell-type specific binding moiety is connected to or attached to the VR8 (protruding) structure. This means that the amino acid sequence for the cell-type specific binding moiety is within the amino acid sequence of VR8. On DNA or RNA level, the nucleotide sequence encoding the cell-type specific binding moiety is located or positioned within the VR8 nucleotide sequence.

[0058] Cell-type specific binding moieties include but is not limited to Affibody molecule, ABD-Derived Affinity Protein (ADAPT), de novo designed proteins, such as asD, TIP-98, TIP-99, GB-CCW9, GBB-CW11, and BAAB-CCW8, or a combination thereof. As shown in example 5, the cell-type specific binding moiety may be located on the same protruding structure, e.g., two cell-type specific binding 84935PC01

[0059] 9 moieties on VR8, or on two different protruding structures, e.g., VR8 and VR4 as demonstrated in example 5.

[0060] Three-helix bundle

[0061] A "three-helix bundle" is a structural motif found in proteins. It consists of three helices, typically alpha helices, connected by loop regions. These helices are packed together in a defined arrangement, usually stabilized by hydrophobic interactions between the side chains of the constituent amino acids. The helices are generally oriented in a roughly parallel or antiparallel fashion, forming a compact and stable core. The N- and C-terminal ends of a three-helix bundle are positioned approximately 180° apart, meaning they point in opposite directions. "Three-helix bundle" and "three-helical bundle" are used interchangeably herein.

[0062] "Monospecific" binding is an interaction where a molecule binds specifically to a single type of marker or receptor. This means the binding molecule has high affinity and specificity for one particular target and does not significantly bind to other molecules. Monospecific binding also applies to bivalent binding molecules.

[0063] "Bispecific" binding is the ability of a molecule to bind to two different markers or receptors, e.g., antigens or epitopes. Thus, the bispecific molecule comprises a dual binding capability for two different targets. Bispecific binding could allow for more precise targeting compared to traditional monospecific binding.

[0064] Bispecific binding is also considered as multi-specific binding.

[0065] In an embodiment of the present invention, the recombinant AAV particle is bispecific. Thus, in an embodiment, the recombinant AAV capsid comprises any combination of cell-type specific binding moiety selected from the group consisting of affibody molecule, designed ankyrin repeat protein (DARPin), nanobody, monobody, affimer, ABD-Derived Affinity Protein (ADAPT), de novo designed proteins, such as a3D, TIP-98, TIP-99, GB-CCW9, GBB-CW11, and BAAB-CCW8, antibody, antibody mimetic, an Fv molecule, an antigen binding fragment, a Fab fragment, a Fab' fragment, and a F(ab')2 molecule, wherein at least one of the cell-type specific binding moieties comprises a three-helix bundle and a length selected from the range of 40 to 150 amino acids.

[0066] The combination of cell-type specific binding moiety can for example be: 84935PC01

[0067] 10

[0068] - two affibodies,

[0069] - two ADAPTS,

[0070] - two asD,

[0071] - an affibody and a nanobody, or

[0072] - an affibody and an affimer.

[0073] Bivalent

[0074] "Bivalent" binding is the ability of a molecule to bind to two identical binding sites simultaneously. The dual binding can enhance the binding, such as stability and / or strength between the bivalent molecule and its target compared to monovalent binding. Bivalent molecules may be considered as monospecific as they have two identical binding sites towards the same target.

[0075] Multi-specific

[0076] "Multi-specific" binding refers to the ability of a molecule to bind to multiple different targets. Multi-specific binding allows for more precise targeting compared to traditional monospecific binding.

[0077] In an embodiment of the present invention, recombinant AAV particle is multispecific. Thus, in an embodiment, the recombinant AAV capsid comprises any combination of cell-type specific binding moiety selected from the group consisting of affibody molecule, designed ankyrin repeat protein (DARPin), nanobody, monobody, affimer, ABD-Derived Affinity Protein (ADAPT), de novo designed proteins, such as a3D, TIP-98, TIP-99, GB-CCW9, GBB-CW11, and BAAB-CCW8, antibody, antibody mimetic, an Fv molecule, an antigen binding fragment, a Fab fragment, a Fab' fragment, and a F(ab')2 molecule, wherein at least one of the cell-type specific binding moieties comprises a three-helix bundle and a length selected from the range of 40 to 150 amino acids. The combination of cell-type specific binding moiety can for example be:

[0078] - two or more affibodies,

[0079] - two or more ADAPTS,

[0080] - two asD,

[0081] - an affibody and a nanobody, or

[0082] - an affibody and an affimer. 84935PC01

[0083] 11

[0084] Affibody molecule

[0085] An affibody molecule is a type of affinity protein comprising a three alpha-helix bundle derived from the Z domain of staphylococcus aureus protein A and consists of only 58 amino acids (approximately 6 kDa), smaller than both DARPins and Nanobodies. Affibody molecules have been developed to have high affinity to a plethora of different targets, including many cell receptors. The affibody molecule is small, low immunogenic, and highly specific.

[0086] In here "affibody molecule" and "affibody" may be used interchangeably.

[0087] Adeno-associated virus (AAV) particle

[0088] An "AAV particle" is a small non-enveloped virus which belongs to the family of Parvoviridae. The AAV genome comprises single stranded DNA of about 4.7 kilo bases. It comprises T-shaped inverted terminal repeats (ITRs) of 145 bases on each end and carries two open reading frames (ORF): Rep and cap, which encode for functional and capsid proteins. The ORF rep encodes for four Rep proteins (Rep78, Rep68, Rep52 and Rep40). Rep 78 and Rep 68 have site-specific, singlestrand endonuclease, DNA helicase, and ATP activities, respectively, which are responsible for DNA replication. Rep52 and Rep40 are responsible for packaging of DNA flanked by ITRs into capsids.

[0089] The ORF cap encodes for three overlapping capsid proteins: VP1, VP2 and VP3. Additionally, the VP1 mRNA encodes for the assembly-activating protein (AAP) in a different reading frame. Based on the differences in the amino acid sequences of the capsid proteins, different AAV serotypes are distinguished, i.e. natural serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAVDJ, AAVrhlO, and AAVrh74. Some AAV serotypes have also been engineered, such as AAV-PHP, AAV-PHP.eB, AAV-LK03, AAV2.5, AAV5.2, AAV-Anc80, and AAV-SparklOO.

[0090] AAV is roughly 20 nm in diameter and comprises capsid proteins including virion proteins (VPs) VP1, VP2 and VP3. Different AAV serotypes have tropism for different tissues, owing to variation in amino acid residues on the AAV capsid surface. The average ratio of VPs in an AAV is 5: 5: 50 of VP1 :VP2:VP3, although capsid VP-composition is more stochastic on individual capsid level. The VPs are encoded from the same gene (Cap gene) with identical C-terminals, but with unique N-terminal regions in VP1 and VP2, which play a role for intracellular 84935PC01

[0091] 12 trafficking upon infection. The single stranded DNA (ssDNA) inside an AAV is flanked by Inverted Terminal Repeats (ITRs).

[0092] In here, an "AAV particle" is to be understood as a composition of:

[0093] - AAV capsid comprising capsid proteins and

[0094] - genetic material, such as a nucleic acid.

[0095] The present invention is not limited to an AAV particle of any specific serotype. Thus, in an embodiment, the AAV serotype is selected from the group consisting of all human AAV serotypes and non-human AAV serotypes, preferably the AAV serotype is a human serotype, more preferably the AAV serotype is human serotypes 2 (AAV2) and 8 (AAV8).

[0096] Recombinant adeno-associated virus

[0097] "Recombinant AAV" as used in the present invention means that the recombinant AAV capsid or particle has been produced by combining genetic material from different sources. Thus, the genes of interest are inserted via a plasmid into a host organism, which uses its own cellular machinery to produce the recombinant AAVs based on the inserted gene.

[0098] The recombinant AAV capsid or particle of the present invention may comprise further modifications in the capsid protein gene for removing the natural tropism of a specific serotype towards a protein. The removal of natural trophism depends on the AAV serotype and their natural degree of tropism towards any cell. Said removal of the natural tropism is not essential for the present invention, however it may be beneficial as it enhances the infectivity of the recombinant AAV particle. For example, as seen in Example 1, the natural tropism of AAV2 towards heparinsulphate proteoglycans (HSPGs) is removed from VP2. Without the removal of HSPG, AAV2 can infect almost any cell with high efficiency. Therefore, the removal of HSPG in the recombinant AAV2 ensures optimal infectivity towards a target cell. Contrary, the natural trophism of AAV8 is unchanged.

[0099] Thus, in an embodiment, the recombinant AAV capsid further comprises removal of natural tropism, such as removal of heparin-sulphate proteoglycan (HSPG) binding motif. In a preferred embodiment, the removal of HSPG is on the engineered virion protein of AAV2. 84935PC01

[0100] 13

[0101] Linker

[0102] The term "linker" refers to a molecule that joins two other molecules, preferably covalently. In the present context, the term "linker" refers to a molecule that joins one end of the cell-type specific binding moiety to a molecule, such as a plasmid or another cell-type specific binding moiety.

[0103] A "linker" is to be understood as a nucleotide linker or DNA linker, which is a short segment of double-stranded DNA.

[0104] DNA linkers serve several purposes in molecular biology and genetic engineering:

[0105] - Connecting DNA Fragments: Linkers are used to join two DNA fragments together, especially when the fragments have blunt ends. This is crucial in cloning and recombinant DNA technology.

[0106] - Adding Restriction Sites: They can introduce specific restriction enzyme sites into DNA sequences. This allows for precise cutting and manipulation of DNA.

[0107] Facilitating Ligation : Linkers help in the ligation process by providing cohesive ends, making it easier to join DNA fragments.

[0108] - Chromatin Structure: In the context of chromatin, linker DNA connects nucleosomes, helping to organize and compact the DNA within the nucleus.

[0109] When the nucleotide linker is translated into protein, it will result in a protein linker, which is a sequence of amino acids within a polypeptide chain that connects distinct protein domains. These linkers typically lack a defined secondary structure, often adopting random coil configurations, which provides flexibility for the overall protein's structure and function. Protein linkers are often used in constructing fusion proteins as they influence the stability and activity of engineered proteins. Protein linkers include but is not limited to a serine glycine linker, a glycine serine linker, or an alanine-proline-serine linker.

[0110] Thus, in an embodiment the cell-type specific binding moiety, such as an affibody molecule, is flanked by a linker, preferably said linker comprises serine and / or glycine, such as a serine glycine linker.

[0111] In a preferred embodiment, the linker comprises an amino acid sequence according to SEQ ID NO. : 59. In another embodiment, the linker comprises a repeat of the amino acid sequence according to SEQ ID NO. : 59. The size of the linker can be the same or different on the N-terminal and C-terminal end of the 84935PC01

[0112] 14 cell-type specific binding moiety. Further, as demonstrated in example 6, a linker can also be present on one side, e.g. the N-terminal side of the cell-type specific binding moiety, and not on the other side of the cell-type specific binding moiety. In a further embodiment, the cell-type specific binding moiety, is flanked by four repeats of the amino acid sequence according to SEQ ID NO. : 59 on the N- terminal end of the cell-type specific binding moiety and five repeats of the amino acid sequence linker according to SEQ ID NO. : 59 on the C-terminal end of the cell-type specific binding moiety.

[0113] As seen in the examples, the affibody molecule is surrounded with serine-glycine linkers to avoid misfolding of VP2. In Z-VP2 with a HER2-binding affibody ZH ER2 :342, the affibody molecule is flanked by the linkers:

[0114] - four S4G N-terminally and

[0115] - five S4G C-terminally

[0116] As seen in example 6 and 7, the affibody-VP2 construct does not necessarily require a linker in order to provide a selective and infective recombinant AAV particle. Thus, in an embodiment, the cell-type specific binding moiety is not flanked by a linker. >

[0117] A "capsid" is the shell of protein of a virus that encloses genetic material. The capsid comprises various proteins, such as virion proteins.

[0118] A "capsid protein" is a component of the protein shell, or capsid, that surrounds and protects the genetic material of a virus.

[0119] Capsid proteins play several crucial roles including protecting the viral genetic material from damage, assisting in attachment of the virus to the target cell, and assisting in delivering viral genetic material to the target cell.

[0120] The AAV capsid proteins comprises different virion proteins.

[0121] Virion protein (VP) M capsid proteins comprises three different overlapping virion proteins (VPs): VP1 (90 kDa), VP2 (72 kDa), VP3 (60 kDa) in the average ratio of 5:5:50. All three VPs are encoded from the same gene (Cap gene), but their initiation start at different positions owing to alternative splicing and alternative start codons. The 84935PC01

[0122] 15

[0123] VPs are identical in their C-terminal, with VP1 being the largest of the three VPs, followed by VP2 and VP3, which are initiated downstream of VP1.

[0124] All three proteins (VP1-VP3) contain a common C-terminal domain of about 530 amino acids and with unique N-terminal regions in VP1 and VP2.

[0125] When referring to any regions or positions in the VPs, VPl-notation is commonly used. Thus, in here, any reference to a position on a nucleic acid or amino acid sequence is with respect to sequence of VP1 according to SEQ ID NO. 1-4.

[0126] For AAV2, VP2 starts at nucleotide position 412-414, amino acid position 138, whereas VP3 starts at nucleotide position 607-609, amino acid position 203 in the Cap gene using VP1 notation. For AAV8, VP2 starts at nucleotide position 412- 414, amino acid position 138, whereas VP3 starts at nucleotide position 634-636, amino acid position 212 in the Cap gene using VP1 notation.

[0127] N-terminus

[0128] The "N-terminus" (or amino-terminus) of a protein is the one end of the protein or polypeptide chain that has a free amine group (-NH2). This is the starting point of the protein.

[0129] Variable region

[0130] The virion proteins comprise a group of conserved strands and several highly diversified interstrand loop regions termed "variable regions" that define the various AAV serotypes. The VPs comprise nine variable regions (VR1-9), which are protruding structures close to or on the capsid surface.

[0131] There are several synonyms for the variable regions:

[0132] • VR4 may also be known as GH2 / GH3 loop

[0133] • VR8 may also be known as GH12 / GH13 loop

[0134] A nucleic acid sequence is an order of nucleotides in a DNA or RNA molecule. These sequences are composed of the nucleotides: Adenine (A), Cytosine (C), Guanine (G), Thymine (T), or Uracil (U).

[0135] The nucleic acid sequence is typically read from the 5' end to the 3' end of the molecule. This nucleic acid sequence encodes genetic information, which is crucial for the functioning and reproduction of living organisms 84935PC01

[0136] 16

[0137] In here, a "nucleic acid" and "nucleic acid sequence" may be used interchangeably.

[0138] Transduction

[0139] In here, "transduction" is a process where foreign DNA is introduced into a target cell by a virus or viral vector.

[0140] Transgene

[0141] A "transgene" according to the present invention is a gene or genetic material that has been transferred from one organism to another. The term "transgene" as used herein describes a segment of a DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism by the gene transfer methods. This non-native segment of DNA may retain the ability to produce RIMA or protein in the transgenic organism. In practical terms, according to the present invention, a transgene can be either a cDNA (complementary DNA) segment, which is a copy of mRNA (messenger RNA), or the gene itself.

[0142] The transgene may be a therapeutic and / or marker / reporter gene, which is packaged into the recombinant AAV particle. More specifically, the transgene is inserted into a vector plasmid and the vector plasmid comprising the transgene is inserted into the host organism when producing the recombinant AAVs.

[0143] In the case that a therapeutic gene and a marker / reporter gene is packaged into the recombinant AAV vector particle, the therapeutic gene and the marker / reporter gene can be inserted into the same vector plasmid.

[0144] A transgene is a gene that is to be transduced into and expressed by the targeted cell. Preferably, the transgene is a therapeutic gene, i.e. a transgene encoding for a therapeutically active product.

[0145] The invention will now be described in further details in the following aspects and embodiments.

[0146] A recombinant adeno-associated virus capsid

[0147] A modular platform for retargeting recombinant AAV particles towards specific cell surface molecules minimizes off-target effects and improves on-target selectivity. Thus, the modular AAV platform of the present invention generates safer AAV treatments with less off-target effects. 84935PC01

[0148] 17

[0149] The inventors have demonstrated improved selectivity and potency of recombinant AAVs comprising at least one cell-type specific binding moiety located on the capsid of the recombinant AAV. In particular, the at least one celltype specific binding moiety is located in variable region 8 (VR8) of a virion protein (VP), thus resulting in an engineered VP (Example 1-3). The fact that the at least one cell-type specific binding moiety is located in a variable region of an AAV capsid virion protein, such as VR8, allows integration of cell-type specific binding moieties across a broader variation of AAV serotypes.

[0150] Thus, an aspect of the invention relates to a recombinant adeno-associated virus (AAV) capsid comprising an engineered virion protein (VP), wherein the engineered VP comprises at least one cell-type specific binding moiety located in variable region 8 (VR8), wherein the cell-type specific binding moiety comprises a three-helix bundle and a length selected from the range of 40 to 150 amino acids. The results in the examples suggest that integration of at least one cell-type specific binding moiety on VR8 provides a more or slightly more specific recombinant AAV capsid compared to integration on VR4. Further, integration of at least one cell-type specific binding moiety on VR8 provides a more potent recombinant AAV capsid compared to integration on the N-terminus. Data show a higher level of integration in VR8 than in VR4 for certain variants.

[0151] Cell-type specific binding moiety in VR8

[0152] A cell-type specific binding moiety targets and binds selectively to specific cell types. These cell-type specific binding moieties achieve specificity through interactions with unique markers or receptors present on the surface of the target cells. It is surprising that the inventors managed to generate recombinant AAV capsids or AAV particles with an affibody (58 amino acids in length) located in VR8 as previous studies have only inserted small peptides of at the most 30 amino acids at said position.

[0153] Thus, in an embodiment, the length of the cell-type specific binding moiety is at least 40 amino acids, more preferably at least 45 amino acids, such as 50 amino acids, most preferably at least 55 amino acids, such as in the range of 40 to 800 amino acids, such as in the range of 40 to 700 amino acids, such as in the range of 40 to 600 amino acids, such as in the range of 45 to 400 amino acids, such as in the range of 45 to 300 amino acids, preferably in the range of 45 to 200 amino acids, more preferably in the range of 45 to 150 amino acids, such as in the range of 50 to 100 amino acids, most preferably in the range of 50 to 80 amino acids. In 84935PC01

[0154] 18 an embodiment, the length of the cell-type specific binding moiety is in the range of 45 to 140 amino acids, such as in the range of 50 to 130 amino acids, such as in the range of 55 to 120 amino acids, such as in the range of 55 to 110 amino acids, such as in the range of 55 to 100 amino acids, preferably in the range of 40 to 90 amino acids, more preferably in the range of 45 to 80 amino acids, most preferably in the range of 50 to 80 amino acids.

[0155] The at least one cell-type specific binding moiety may be any type of cell-type specific binding moiety having a length within the above listed ranges. As seen in Example 1-5, different Affibody molecules were successfully integrated on VR8 in AAVs of different serotypes. An Affibody molecule comprises 58 amino acids (6 kDa), thus the length of an Affibody molecule falls within the above ranges.

[0156] The size of the cell-type specific binding moiety may also be expressed in molecular weight in kilodalton (kDa). In an embodiment, the size of the cell-type specific binding moiety is at least 4 kDa, such as at least 4.5 kDa, preferably at least 5 kDa, more preferably at least 5.5 kDa, most preferably at least 6 kDa, such as in the range of 4 kDa to 80 kDa, such as in the range of 4 kDa to 70 kDa, such as in the range of 5 kDa to 60 kDa, such as in the range of 5 kDa to 50 kDa, preferably in the range of 5.5 kDa to 40 kDa, such as in the range of 5.5 kDa to 30 kDa, more preferably in the range of 6 kDa to 20 kDa, most preferably in the range of 6 kDa to 10 kDa.

[0157] In an embodiment, the cell-type specific binding moiety is selected from the group consisting of Affibody molecule, designed ankyrin repeat protein (DARPin), nanobody, monobody, affimer, ABD-Derived Affinity Protein (ADAPT), antibody, antibody mimetic, an Fv molecule, an antigen binding fragment, a Fab fragment, a Fab' fragment, and a F(ab')? molecule, or a combination thereof. In an embodiment, the three-helix bundle is a three alpha-helix bundle. In another embodiment, the cell-type specific binding moiety is selected from the group consisting of Affibody molecule, ABD-Derived Affinity Protein (ADAPT), de novo designed proteins, such as asD, TIP-98, TIP-99, GB-CCW9, GBB-CW11, and BAAB-CCW8, or a combination thereof.

[0158] Thus, the at least one cell-type specific binding moiety located in VR8 may be any of the binding moieties listed above. 84935PC01

[0159] 19

[0160] As seen in the Examples, the inventors have successfully demonstrated that the cell-type specific binding moiety could be an Affibody molecule. Said recombinant AAV capsid comprising an Affibody molecule located in VR8 demonstrates selectivity and selective infection towards a specific target. Thus, in an embodiment, the cell-type specific binding moiety is an Affibody molecule.

[0161] In a preferred embodiment, the Affibody molecule is selected from the group consisting of SEQ ID NO. 33 to 44, and a combination thereof. The SEQ ID NOs. 33-44 are the sequences of Affibody molecules which have been used in the Examples.

[0162] Additional cell-type specific binding moiety

[0163] The recombinant AAV capsid according to the present invention may comprise more than one cell-type specific binding moiety. In a situation where the recombinant AAV capsid comprises more than one cell-type specific binding moiety, at least one of the cell-type specific binding moieties is located in VR8. In an embodiment, the recombinant AAV capsid further comprises at least one additional cell-type specific binding moiety.

[0164] The integration of at least one additional cell-type specific binding moiety in addition to the cell-type specific binding moiety attached to VR8, results in a recombinant AAV capsid with enhanced target binding, such as enhanced stability and / or strength between the cell-type specific binding moiety and target and allows for more precise targeting.

[0165] Thus, in an embodiment, the at least one additional cell-type specific binding moiety is identical to or different from the cell-type specific binding moiety located in VR8.

[0166] The additional cell-type specific binding moiety may be identical or different from the cell-type specific binding moiety located in VR8 in terms of type and / or targeting. The type of the additional cell-type specific binding moiety may be identical or different from the cell-type specific binding moiety located in VR8. The term "type" used in this context refers to a category of cell-type specific binding moieties. The cell-type specific binding moiety include but is not limited to Affibody molecule, designed ankyrin repeat protein (DARPin), nanobody, monobody, affimer, ABD-Derived Affinity Protein (ADAPT), de novo designed proteins, such as a3D, TIP-98, TIP-99, GB-CCW9, GBB-CW11, and BAAB-CCW8, 84935PC01

[0167] 20 antibody, antibody mimetic, an Fv molecule, an antigen binding fragment, a Fab fragment, a Fab' fragment, and a F(ab')? molecule, or a combination thereof.

[0168] Thus, in an embodiment, the at least one additional cell-type specific binding moiety is selected from the group consisting of Affibody molecule, designed ankyrin repeat protein (DARPin), nanobody, monobody, affimer, ABD-Derived Affinity Protein (ADAPT), de novo designed proteins, such as asD, TIP-98, TIP-99, GB-CCW9, GBB-CW11, and BAAB-CCW8, antibody, antibody mimetic, an Fv molecule, an antigen binding fragment, a Fab fragment, a Fab' fragment, and a F(ab')2 molecule, or a combination thereof.

[0169] The combination of cell-type specific binding moieties on the recombinant AAV capsid may for example be:

[0170] - two or more affibodies,

[0171] - two or more ADAPTS,

[0172] - two asD,

[0173] - an affibody and a nanobody, or

[0174] - an affibody and an affimer.

[0175] As seen in Example 5, a bispecific recombinant AAV capsid and a bivalent recombinant AAV capsid have been constructed comprising one Affibody molecule located in VR8 and one Affibody molecule located in VR4. Thus, in an embodiment, the at least one additional cell-type specific binding moiety is an Affibody molecule. The bispecific or bivalent recombinant AAV capsid presents enhanced target binding, such as enhanced stability and / or strength, compared to monovalent or monospecific recombinant AAV capsids, and allows for more precise targeting.

[0176] In a preferred embodiment, the Affibody molecule is selected from the group consisting of SEQ ID NO. 33-44, and a combination thereof. The SEQ ID NOs. 33- 44 are the sequences of Affibody molecules which have been used in the Examples.

[0177] In an embodiment, the at least one additional cell-type specific binding moiety is located on / in a protruding structure of the recombinant AAV capsid, such as a protruding loop, an alpha-helix, or a beta-sheet. In an embodiment, the protruding structure of the recombinant AAV capsid is selected from the group 84935PC01

[0178] 21 consisting of protruding loop, alpha-helix, and beta-sheet, or a combination thereof. The at least one additional cell-type specific binding moiety is attached to a protruding structure to ensure that the cell-type specific binding moiety is accessible to bind a target cell.

[0179] Protruding structures are located in several regions in the AAV capsid virion protein, thus in principle, the at least one additional cell-type specific binding moiety may be attached to any protruding structure of the AAV capsid virion protein, preferably provided that such attachment allows the at least one additional cell-type specific binding moiety being positioned on the surface of the recombinant AAV capsid. In an embodiment, the protruding structure is located at a position selected from the group consisting of N-terminus, variable region 1 (VR1), variable region 4 (VR4), variable region 5 (VR5), variable region 6 (VR6), between VR5 and VR6, variable region 7 (VR7), and variable region 8 (VR8), preferably VR4. The entire region from VR5 to VR6, including the amino acids between VR5 and VR6, are part of a protruding structure on the AAV capsid. Hence, based on structural studies the inventors believe that a cell-type specific binding moiety could be grafted anywhere in the region of VR5 to VR6.

[0180] The at least one additional cell-type specific binding moiety may be located in VR8 together with the cell-type specific binding moiety already attached on VR8, resulting in a recombinant AAV capsid comprising two or more cell-type specific binding moieties on VR8. In addition, the at least one additional cell-type specific binding moiety may be located in a different position than VR8, such as N- terminus and / or VR4. As seen in Example 5, the bispecific or bivalent recombinant AAV capsid comprises one cell-type specific binding moiety on VR8 and one additional cell-type specific binding moiety on VR4. Especially, the bispecific recombinant AAV capsid enables robust AAV-cancer treatments to avoid loss-of- function in the case of phenotype switching as seen in Example 5.

[0181] In terms of targeting of the cell-type specific binding moieties, the inventors have also generated a recombinant AAV capsid with two cell-type specific binding moieties on two different insertion points for bispecific or bivalent AAV targeting. Thus, in an embodiment, the recombinant AAV capsid is monospecific or multispecific, such as bispecific or bivalent. In a further embodiment, the recombinant AAV capsid is monovalent or multivalent, such as bivalent. 84935PC01

[0182] 22

[0183] If the targeting of the at least one additional cell-type specific binding moiety is identical to the cell-type specific binding moiety located in VR8, the resulting recombinant AAV capsid is multivalent, more specifically bivalent. The bivalency of the recombinant AAV capsid enhances the interaction between the bivalent recombinant AAV capsid and its target cell compared to monovalent / monospecific recombinant AAV capsid. Hence, in an embodiment, the recombinant AAV capsid is bivalent.

[0184] If the targeting of the at least one additional cell-type specific binding moiety is different from the cell-type specific binding moiety located in VR8, the resulting recombinant AAV capsid is multi-specific, such as bispecific. Multi-specific binding of the recombinant AAV capsid allows for more precise targeting compared to traditional monospecific binding. When a tumour cell is treated by targeting a specific receptor, the cancer sometimes switches phenotype and upregulates other receptors to compensate. To combat phenotype switching affecting the infectivity of mono-specific AAV capsids, targeting two or more different cell receptors at the same time using multi-specific, such as bispecific AAV capsids could be beneficial. The inventors successfully generated bi-specific AAV capsids in Example 5. Hence, in an embodiment, the recombinant AAV capsid is bispecific.

[0185] One or more surface receptors

[0186] Cell-type specific binding moieties are useful in targeting and binding to specific cell types. These binding moieties achieve specificity through interactions with markers and / or receptors present on the surface of target cells. In an embodiment, the cell-type specific binding moiety is specific for one or more surface receptors expressed on a target cell.

[0187] In another embodiment, the one or more surface receptors are selected from the group consisting of ErbB receptor family, insulin receptor family, platelet-derived growth factor receptor (PDGFR) family, vascular endothelial growth factor receptor (VEGFR) family, transferrin receptor family, and Ly6 receptor family, or a combination thereof, preferably ErbB receptor family, insulin receptor family, platelet-derived growth factor receptor (PDGFR) family, or vascular endothelial growth factor receptor (VEGFR) family, more preferably ErbB receptor family, or insulin receptor family. As seen in the Examples, the recombinant AAV capsid of the invention is specific towards HER2, HER3, IGF1R, EGFR, PDGFRp, and / or VEGFR2. Thus, in a preferred embodiment, the one or more surface receptors are 84935PC01

[0188] 23 selected from the group consisting of HER2, HER3, EGFR, IGF1R, PDGFRb, and VEGFR2, preferably HER2, HER3, EGFR, or IGF1R, more preferably HER2, HER3, or EGFR.

[0189] Target cell

[0190] Surface receptors are found on the surface of cells. Specific cells may comprise a unique composition of surface receptors making specific surface receptors ideal targets for binding a specific cell. In principle, the cell may be any type of target cell comprising surface receptors. However, in an embodiment, the target cell is a mammalian cell. As seen in the examples, the recombinant AAV capsid of the invention indeed works for mammalian cells.

[0191] In an embodiment, the mammalian cell is selected from the group consisting of human cell, mouse cell, rat cell, cynomolgus monkey cell, rhesus monkey cell, cattle cell, pig cell, horse cell, sheep cell, goat cell, mink cell, hamster cell, cat cell, and dog cell. As presented in the examples, the recombinant AAV capsid of the invention indeed works for human cells. Thus, in a preferred embodiment, the mammalian cell is a human cell.

[0192] The linker

[0193] The cell-type specific binding moiety may be surrounded by a linker. Thus, in an embodiment, the cell-type specific binding moiety is flanked by a linker.

[0194] As seen in the examples, the cell-type specific binding moiety, such as the affibody molecule, is surrounded with serine-glycine linkers on both the N- terminal and C-terminal end to stabilize and avoid misfolding of the engineered VP.

[0195] In principle, the linker may be any flexible linker comprising small amino acids. For example, the cell-type specific binding moiety is flanked by a linker comprising serine and / or glycine. In an embodiment, the linker comprises serine and / or glycine.

[0196] In another embodiment, the linker is a serine glycine linker, a glycine serine linker, or an alanine-proline-serine linker, preferably a repeat of the serine glycine linker according to SEQ ID NO. : 59. In a preferred embodiment, the linker comprises an amino acid sequence according to SEQ ID NO. : 59. In another embodiment, the linker comprises a repeat of the amino acid sequence according to SEQ ID NO. : 59. 84935PC01

[0197] 24

[0198] In the examples, the cell-type specific binding moiety, is flanked by four repeats of the amino acid sequence according to SEQ ID NO. : 59 on the N-terminal end of the cell-type specific binding moiety and five repeats of the amino acid sequence linker according to SEQ ID NO. : 59 on the C-terminal end of the cell-type specific binding moiety. In a further embodiment, the cell-type specific binding moiety, is flanked by four repeats of the amino acid sequence according to SEQ ID NO. : 59 on the N-terminal end of the cell-type specific binding moiety and five repeats of the amino acid sequence linker according to SEQ ID NO. : 59 on the C-terminal end of the cell-type specific binding moiety.

[0199] Further, the size or length of the linker can be the identical or different on the N- terminal and C-terminal end of the cell-type specific binding moiety. As demonstrated in example 6, a linker can be present on one side, e.g. the N- terminal side of the cell-type specific binding moiety, and not on the other side of the cell-type specific binding moiety.

[0200] It has been demonstrated that a linker is not required for the functionality of the recombinant AAV capsid according to the invention.

[0201] Thus, in a further embodiment, the cell-type specific binding moiety is not flanked by a linker. In Example 6, it is observed that the cell-type specific binding moiety, such as the affibody molecule, without being flanked by any linkers, results in a well-performing recombinant AAV capsid or particle. The absence of linkers does not adversely affect the selectivity and infectivity of the recombinant AAV capsid or particle.

[0202] VR8 position

[0203] In an embodiment, the VR8 is located in the engineered virion protein (VP) between amino acid position 560 and 610 relative to SEQ ID NO.: 2 or 4. SEQ ID NOs. 2 and 4 are the sequences of VP1 of AAV2 and AAV8, respectively.

[0204] In an embodiment, the cell-type specific binding moiety is located in VR8 after amino acid position 587 relative to SEQ ID NO. : 2 or amino acid position 590 relative to SEQ ID NO. : 4. SEQ ID NOs. 2 and 4 are the sequences of VP1 of AAV2 and AAV8, respectively. 84935PC01

[0205] 25

[0206] VR4 position

[0207] In an embodiment, the VR4 is located in the engineered virion protein (VP) between amino acid position 430 and 480 relative to SEQ ID NO.: 2 or 4. SEQ ID NOs. 2 and 4 are the sequences of VP1 of AAV2 and AAV8, respectively.

[0208] In an embodiment, the cell-type specific binding moiety is located in VR4 after amino acid position 452 relative to SEQ ID NO. : 2 or amino acid position 455 relative to SEQ ID NO. : 4. SEQ ID NOs. 2 and 4 are the sequences of VP1 of AAV2 and AAV8, respectively.

[0209] Enqineered VP

[0210] In an embodiment, the engineered VP is derived from a VP selected from the group consisting of virion protein 1 (VP1), virion protein 2 (VP2), and virion protein 3 (VP3), preferably VP1 or VP2, more preferably VP2. In another embodiment, the engineered VP is selected from the group consisting of an engineered virion protein 1 (VP1), engineered virion protein 2 (VP2), and engineered virion protein 3 (VP3), preferably engineered VP1 or engineered VP2, more preferably engineered VP2. In Examples 1-6, the engineered VP is derived from VP2.

[0211] In principle, the recombinant AAV capsid may be derived from any AAV serotype. Thus, in an embodiment, the AAV is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAVDJ, AAVrhlO, AAVrh74, AAV-PHP, AAV-PHP.eB, AAV-LK03, AAV2.5, AAV5.2, AAV-Anc80, and AAV-SparklOO, preferably AAV2, AAV5, AAV8 and AAV9, more preferably AAV2 or AAV8. As seen in the examples, the recombinant AAV capsid can be derived from AAV2 and AAV8.

[0212] In an embodiment, the engineered VP comprises the amino acid sequence according to SEQ ID NO. 24 or 28.

[0213] A recombinant adeno-associated virus (AAV) particle

[0214] The recombinant AAV capsid according to the invention is included in a recombinant AAV particle, which may be used to infect target cells.

[0215] Another aspect of the invention relates to a recombinant adeno-associated virus (AAV) particle comprising the recombinant AAV capsid according to the present 84935PC01

[0216] 26 invention. As can be seen in the examples, recombinant AAV particles comprising recombinant AAV capsid are isolated and applied to target proteins and target cells.

[0217] In principle, the recombinant AAV capsid may be derived from any AAV serotype. In an embodiment, the AAV is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAVDJ, AAVrhlO, AAVrh74, AAV-PHP, AAV-PHP.eB, AAV-LK03, AAV2.5, AAV5.2, AAV-Anc80, and AAV-SparklOO, preferably AAV2, AAV5, AAV8 and AAV9, more preferably AAV2 or AAV8. As seen in the examples, the recombinant AAV capsid can be derived from AAV2 and AAV8.

[0218] In the proof-of-concept experiment conducted in Example 4, the inventors demonstrated that the AAV particles of the present invention could specifically target and hinder tumour cell proliferation. In a clinical setting, the recombinant AAV particles would comprise a nucleic acid. Thus, in an embodiment, the recombinant AAV particle further comprises at least one nucleic acid. The recombinant AAV particle could also comprise more than one nucleic acid, e.g., the nucleic acid could treat the same disease by having multiple nucleic acids encoding different therapeutic active products.

[0219] In an embodiment, the nucleic acid is selected from the group consisting of singlestranded DNA (ssDNA), self-complementary DNA (scDNA), messenger RIMA (mRNA), self-amplifying RNA, circular RNA (circRNA), plasmid DNA (pDNA), small interfering RNA (siRNA), single guide RNA (sgRNA), guide RNA (gRNA), long noncoding RNA (IncRNA), small activating RNA (saRNA), and splice-switching antisense oligonucleotide (ASO), or a combination thereof. In principle, the nucleic acid may be any nucleic acid which can be packaged into the recombinant AAV particle for delivery to a target cell. AAVs are ssDNA viruses, hence the ssDNA can be packaged as normal ssDNA or as scDNA depending on the design of the nucleic acid.

[0220] In a preferred embodiment, the nucleic acid is a transgene. In principle, the transgene may be any transgene. For example, the transgene may be herpes simplex virus thymidine kinase (HSV-TK) as shown in Example 4. 84935PC01

[0221] 27

[0222] In a preferred embodiment, the nucleic acid encodes a therapeutically active product.

[0223] A system

[0224] As mentioned above, the recombinant AAV particle of the invention can be utilized to deliver cargo, such as a nucleic acid, to a target cell. Thus, a further aspect of the invention relates to a system for delivering at least one nucleic acid to a cell determined by a cell-type specific binding moiety, said system comprises a recombinant adeno-associated virus (AAV) particle according to the invention. The recombinant AAV particle comprises a recombinant AAV capsid, wherein the recombinant AAV capsid comprises at least one cell-type specific binding moiety, hence, the recombinant AAV particle is capable of selectively targeting a cell of interest. With the addition of nucleic acid in the recombinant AAV particle, the recombinant AAV particle of the invention will selectively bind to a target cell and deliver said nucleic acid to said target cell.

[0225] In an embodiment, the nucleic acid is selected from the group consisting of singlestranded DNA (ssDNA), self-complementary DNA (scDNA), messenger RIMA (mRNA), self-amplifying RNA, circular RNA (circRNA), plasmid DNA (pDNA), small interfering RNA (siRNA), single guide RNA (sgRNA), guide RNA (gRNA), long noncoding RNA (IncRNA), small activating RNA (saRNA), and splice-switching antisense oligonucleotide (ASO), or a combination thereof. In principle, the nucleic acid may be any nucleic acid which can be packaged into the recombinant AAV particle for delivery to a target cell.

[0226] In an embodiment, the nucleic acid is a transgene. In principle, the transgene may be any transgene. For example, the transgene may be herpes simplex virus thymidine kinase (HSV-TK) as shown in Example 4.

[0227] In an embodiment, the nucleic acid encodes a therapeutically active product.

[0228] A genetic construct

[0229] An aspect of the invention relates to a genetic construct comprising a nucleotide sequence encoding a recombinant AAV capsid comprising at least one cell-type specific binding moiety located in variable region 8 (VR8). Another aspect of the 84935PC01

[0230] 28 invention relates to a genetic construct comprising a nucleotide sequence encoding a recombinant AAV capsid according to the invention.

[0231] A genetic construct may be any construct for delivering and transferring genes and / or nucleotide sequences to a host cell.

[0232] Another aspect of the invention relates to a plasmid comprising a nucleotide sequence encoding a recombinant AAV capsid comprising at least one cell-type specific binding moiety located in variable region 8 (VR8).

[0233] A further aspect of the invention relates to a doggybone DNA (dbDNA) comprising a nucleotide sequence encoding a recombinant AAV capsid comprising at least one cell-type specific binding moiety located in variable region 8 (VR8).

[0234] Yet another aspect of the invention relates to a genetic construct comprising a nucleotide sequence encoding an engineered virion protein (VP) comprising a celltype specific binding moiety located in variable region 8 (VR8).

[0235] Yet another aspect of the invention relates to a plasmid comprising a nucleotide sequence encoding an engineered virion protein (VP) comprising a cell-type specific binding moiety located in variable region 8 (VR8).

[0236] Yet another aspect of the invention relates to a doggybone DNA (dbDNA) comprising a nucleotide sequence encoding an engineered virion protein (VP) comprising a cell-type specific binding moiety located in variable region 8 (VR8).

[0237] In an embodiment, the genetic construct comprises a restriction site. In another embodiment, the restriction site is selected from the group consisting of Ascl / Notl, and BamHI / Xmnl.

[0238] In an embodiment, the nucleotide sequence encoding an engineered virion protein (VP) comprising a cell-type specific binding moiety located in variable region 8 (VR8) is inserted into the genetic construct using Ascl / Notl restriction site. 84935PC01

[0239] 29

[0240] In a further embodiment, the cell-type specific binding moiety is inserted into the genetic construct using a restriction site. In another embodiment, said restriction site is BamHI / Xmnl.

[0241] In an embodiment, the genetic construct is selected from the group consisting of plasmid, messenger RIMA (mRNA), doggybone DNA (dbDNA), yeast artificial chromosome (YAC), and bacterial artificial chromosome (BAC).

[0242] Medical use

[0243] It has been demonstrated that the recombinant AAV particle according to the invention can be applied as a medicament and for use in the treatment of diseases. In particular, it is shown in Example 4 that the recombinant AAV particle according to the invention is able to selectively deliver a gene to a target cell, hence the recombinant AAV particle or the system comprising the recombinant AAV particle according to the invention holds great potential for therapeutic application. Thus, an aspect of the invention relates to the recombinant AAV particle according to the present invention or the system according to the present invention for use as a medicament.

[0244] 2ndmedical use

[0245] Example 4 shows a proof-of-concept for the use of the recombinant AAV particle or the system comprising the recombinant AAV particle according to the invention in the treatment of diseases. Particularly, it is shown in Example 4 that the recombinant AAV particle of the invention can specifically target and hinder cancer cell proliferation. In principle, the recombinant AAV particle or the system of the invention can be applied for use in the treatment, alleviation, and / or prevention of any disease. As shown in Example 3, the recombinant AAV particle of the invention can be retargeted towards any target marker or receptor and thereby any target cell of interest. Hence, any target cell responsible for a specific disease can be subjected to the recombinant AAV particle or the system of the invention. A further aspect of the invention relates to the recombinant AAV particle according to the present invention or the system according to the present invention for use in the treatment, alleviation, and / or prevention of a disease selected from the group consisting of cancer, Alzheimer's disease, Parkinson's disease, epilepsy, Autosomal dominant polycystic kidney disease (ADPKD), Nephronophthisis (NPHP), lipoprotein lipase deficiency, Leber's congenital amaurosis, Spinal muscle 84935PC01

[0246] 30 atrophy (SMA), Haemophilia A, Haemophilia B, Duchenne muscular dystrophy (DMD), and Aromatic L-amino acid decarboxylase (AADC) deficiency, preferably cancer.

[0247] In an embodiment, the cancer is selected from the group consisting of gastric cancer, breast cancer, leukemia, such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic myelocytic granulocytic leukemia, chronic lymphocytic leukemia, lymphoma, such as Hodgkin's disease and non-Hodgkin's disease, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, colon carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hepatoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, oligodendroglioma, melanoma, neuroblastoma, retinoblastoma, dysplasia and hyperplasia, prostate cancer, and prostate adenocarcinoma, preferably gastric cancer or breast cancer. The recombinant AAV particle of the present invention was applied in the proof-of- concept cancer assay, where the recombinant AAV particle selectively and significantly reduced the proliferation of cancer cells (Example 4), thus the recombinant AAV particle of the invention is able to specifically target and hinder cancer cell proliferation. The cancer cell lines used in the examples were NCI-N87, SK-BR-3, and MCF-7. MCF-7 and SK-BR-3 are both breast cancer cell lines, whereas NCI-N87 is a gastric cancer cell line. The results demonstrate a potential use of the platform in the treatment, alleviation, and / or prevention of diseases, such as in cancer treatment.

[0248] In principle, the recombinant AAV particle or system of the present invention can be administered via any administration route suitable for delivery of a functional recombinant AAV particle. In an embodiment, the recombinant AAV particle or the system is administered to a subject via an administration route selected from the group consisting of intravenous, intradermal, intramuscular, subcutaneous, convection-enhanced delivery, intranasal, intrathecal, intracerebral, transurethral, retrourethral, and subretinal. 84935PC01

[0249] 31

[0250] In an embodiment, the subject is a mammal. As seen in the examples, the recombinant AAV particle or the system of the invention indeed works for mammalian cells. In another embodiment, the mammal is selected from the group consisting of human, mouse, rat, cynomolgus monkey, rhesus monkey, cattle, pig, horse, sheep, goat, mink, hamster, cat, and dog. As presented in the examples, the recombinant AAV particle or system of the invention indeed works for human cells. Thus, in a preferred embodiment, the mammalian is a human.

[0251] Method for producing the AAV particle

[0252] An aspect of the invention relates to a method for producing the recombinant AAV particle according to the present invention, said method comprising the steps: al) providing a first genetic construct comprising a nucleotide sequence encoding an engineered virion protein (VP) comprising a cell-type specific binding moiety located in variable region 8 (VR8), or a2) providing a first genetic construct encoding an adeno-associated virus (AAV), wherein the start codon of a virion protein (VP) is mutated, and providing a second genetic construct comprising a nucleotide sequence encoding an engineered VP comprising at least one cell-type specific binding moiety located in variable region 8 (VR8), b) transfecting the first genetic construct of step al) or the first genetic construct and the second genetic construct of step a2) into host cells in conditions suitable for said host cells to produce AAV particles, and c) isolating AAV particles, wherein the mutated VP and the engineered VP are the same VP variant.

[0253] The VP variant is to be understood as one of the virion proteins VP1, VP2, or VP3. Thus, in an embodiment, the VP variant is selected from the group consisting of virion protein 1 (VP1), virion protein 2 (VP2), and virion protein 3 (VP3), preferably VP1 or VP2, more preferably VP2. As seen in Example 1, the mutated VP and the engineered VP are both VP2.

[0254] In an embodiment, the genetic construct encoding an adeno-associated virus (AAV) comprises a Rep gene and a Cap gene. 84935PC01

[0255] 32

[0256] In an embodiment, the host cells are additionally transfected with a genetic construct comprising at least one helper gene and / or a genetic construct comprising at least one nucleic acid. A genetic construct comprising at least one nucleic acid may be a nucleic acid for delivery to a target cell.

[0257] In the context of viral vectors, such as AAVs, the helper genes assist in the replication and packaging of the viral genome. These genes help in the production of infectious viral particles. In an embodiment, the helper gene is selected from the group consisting of E4, E2a and VA-RNA, or a combination thereof, preferably a combination of E4, E2a and VA-RNA.

[0258] In an embodiment, the nucleic acid is a transgene. The transgene may be herpes simplex virus thymidine kinase (HSV-TK) as shown in Example 4.

[0259] In an embodiment, the genetic construct is selected from the group consisting of plasmid, messenger RNA (mRNA), doggybone DNA (dbDNA), yeast artificial chromosome (YAC), and bacterial artificial chromosome (BAC). In principle, the genetic construct may be any construct for delivering and transferring genetic material into a host cell for expression.

[0260] In an embodiment, the AAV is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAVDJ, AAVrhlO, AAVrh74, AAV-PHP, AAV-PHP.eB, AAV-LK03, AAV2.5, AAV5.2, AAV-Anc80, and AAV-SparklOO, preferably AAV2, AAV5, AAV8 and AAV9, more preferably AAV2 or AAV8. In principle, the recombinant AAV particle may be derived from any AAV serotype. As seen in the examples, the recombinant AAV capsid can be derive from AAV2 and AAV8.

[0261] In an embodiment, the mutation of the start codon of the virion protein (VP) of step a2) is a point mutation. In a preferred embodiment, the mutation of the start codon of virion protein (VP) of step a2) is performed by site-directed mutagenesis. As seen in Example 1, the start codon of VP2 in a plasmid encoding AAV is performed to remove wildtype VP2 expression. The start codon of VP2 in a RepCap plasmid was mutated through site-directed mutagenesis. Afterwards, the 84935PC01

[0262] 33

[0263] VP2 expression was reintroduced via a second plasmid comprising the engineered VP2 comprising the cell-type specific binding moiety of interest.

[0264] In an embodiment, the cell-type specific binding moiety is specific for one or more targets selected from the group consisting of ErbB receptor family, insulin receptor family, platelet-derived growth factor receptor (PDGFR) family, vascular endothelial growth factor receptor (VEGFR) family, transferrin receptor family, and Ly6 receptor family, preferably ErbB receptor family, insulin receptor family, platelet-derived growth factor receptor (PDGFR) family, or vascular endothelial growth factor receptor (VEGFR) family. As seen in the Examples, the recombinant AAV particle was specific towards HER2, HER3, IGF1R, EGFR, PDGFRp, and / or VEGFR2. Thus, in a preferred embodiment, the cell-type specific binding moiety is specific for one or more targets selected from the group consisting of HER2, HER3, EGFR, IGF1R, PDGFRb, and VEGFR2.

[0265] Alternative aspects

[0266] An alternative aspect of the present invention is to provide an engineered adeno- associated virus (AAV) capsid protein comprising at least one cell-type specific binding moiety located in variable region 8 (VR8).

[0267] Another alternative aspect of the present invention relates to a method for treating or alleviating a subject in the need thereof, the method comprising administering to the subject a composition comprising at least one recombinant adeno-associated virus (AAV) particle comprising an engineered virion protein (VP), wherein the engineered VP comprises at least one cell-type specific binding moiety located in variable region 8 (VR8).

[0268] An alternative aspect relates to a recombinant adeno-associated virus (AAV) capsid comprising an engineered virion protein (VP), wherein the engineered VP comprises at least one cell-type specific binding moiety attached to or connected to variable region 8 (VR8), wherein the cell-type specific binding moiety comprises a three-helix bundle and a length selected from the range of 40 to 150 amino acids.

[0269] Another alternative aspect relates to a recombinant adeno-associated virus (AAV) capsid comprising an engineered virion protein (VP), wherein the engineered VP 84935PC01

[0270] 34 comprises at least one cell-type specific binding moiety fused onto variable region 8 (VR8), wherein the cell-type specific binding moiety comprises a three-helix bundle and a length selected from the range of 40 to 150 amino acids.

[0271] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0272] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0273] The invention will now be described in further details in the following non-limiting examples.

[0274] Examples f recombinant adeno-associated virus molecule

[0275] Aim of study

[0276] The aim of the study was to integrate affibody molecules (Z) in AAVs to create a modular platform to re-target AAVs to specific cells or cell receptors for selective delivery of DNA.

[0277] Materials and methods

[0278] Cells culture

[0279] The production of AAVs was done in 293T cells (CRL-3216, ATCC), grown in Dulbecco's Modified Eagle Medium (41966029, Gibco) supplemented with 10% Heat-inactivated Foetal Bovine Serum (16140071, Gibco or S140H-500, Biowest). Cells were grown in 37°C, 5% CO2. ExpiCHO cells (A29127, Gibco) were maintained in ExpiCHO Expression Media (A2910001, Gibco) and grown at 37°C, 5% CO2, 120 rpm. In the cancer cell assays, NCI-N87 (CRL-5822, ATCC), SK-BR- 3 (HTB-30, ATCC), MCF-7 (HTB-22, ATCC) and hDFn (C0045C, Gibco) were used. NCI-N87 were maintained in RPMI1640 (21875034, Gibco) + 10 % HI FBS (S140H-500, Biowest), SK-BR-3 in Modified McCoy 5A (16600082, Gibco) + 10 % 84935PC01

[0280] 35

[0281] HI FBS, MCF-7 in Dulbecco's Modified Eagle Medium (41966029, Gibco) + 10% HI FBS and hDFn in DMEM / F-12 (10565018, Gibco) + 5 % HI FBS. All cells were maintained at 37°C, 5% CO2.

[0282] Helper plasmid (encoding E4, E2a and VA-RNA), eGFP (green fluorescent protein) cargo and RepCap (Rep gene and Cap gene) for AAV2 were cloned into the pKTH16small minimal vector. Subsequently, AAV2 cap was exchanged with AAV8 cap through HiFi Cloning according to manufacturer's recommendations (E2621L, New England Biolabs). HSV-TK from Human Herpes simplex virus 1 (UniProt: Q9QNF7) was ordered as a gBIock (Integrated DNA Technologies) flanked by Ascl / Notl sites, exchanging the eGFP sequence in the cargo plasmid through restriction cloning.

[0283] To remove wildtype VP2 expression, the start codon of VP2 in AAV2 and AAV8 RepCap plasmid was mutated through site-directed mutagenesis with primers AAV2 dVP2 fwd, AAV2 dVP2 rev, AAV8 dVP2 fwd and AAV8 dVP2 rev (SEQ ID NO. : 47-50).

[0284] For AAV8, an additional ACG codon 57 bp downstream of the commonly annotated start codon could preferably be mutated to ACA to fully remove wildtype VP2 expression (primers AAV8 2nd dVP2 fwd and AAV8 2nd dVP2 rev (SEQ ID NO. : 51 and 52)). Site-directed mutagenesis was also used to remove the HSPG-binding motif in AAV2 (primers dHSPG fwd and dHSPG rev (SEQ ID NO. : 45 and 46)).

[0285] Z-VP2 with a HER2-binding Affibody flanked by linkers 4x S4G (SEQ ID NO. : 59) N-terminally (VR4 and VR8 variants) and 5x S4G C-terminally (all variants) were ordered as gBIocks (IDT). The Z-construct was inserted after amino acid (aa) 452 for VR4 Z-AAV2 while also removing aa 453-459. For VR4 Z- AAV8, the same construct was inserted after aa 455, removing aa 456-462. The Z-construct was inserted after aa 587 for VR8 Z-AAV2 and aa 590 for VR8 Z-AAV8 with no removal of amino acids. The Z-VP2 construct was cloned into the pKTH16small vector using restriction cloning (Ascl / Notl). To modularly exchange affibody molecules in the VR8 Z-VP2 for AAV2, BamHI / XmnI restriction cloning was used. The affibody molecule was exchanged for one of the following: ZvEGFR2_16matG-40matB-40-PROSS6) . 84935PC01

[0286] 36

[0287] For the BiZ-AAV2s, the Z-construct from VR8 was amplified by PCR (primers VR4 Backbone - fwd at VR8 site, VR4 Backbone - rev at VR8 site (SEQ ID NO. : 55 and 56)) and cloned into the PCR-amplified VR4 Z-VP2 vector (VR8 Z amplification fwd, VR8 Z amplification rev (SEQ ID NO. : 57 and 58)) using HiFi cloning to create a BiZ-VP2 vector with affibody molecules on both VR4 and VR8.

[0288] Z-AAV8 with varied linker lengths were cloned by amplifying the VR8 Z-VP2 AAV8 by PCR (primers AAV8 VR8 fwd (SEQ ID NO. 60), AAV8 VR8 rev (SEQ ID NO. 61)) and subsequent HiFi cloning of gBIocks of ZHER2:342 surrounded by varying linker lengths. Z-AAV2 with reduced or no linkers were cloned using HiFi cloning, gBIocks and aforementioned primers.

[0289] Plasmid production

[0290] Plasmids were purified from overnight Tryptic Soy Broth cultures supplemented with 50 pg / ml Kanamycin, cultivated at 37°C, 150 rpm. An in-house strain of ToplO was used for all plasmids except for Cargo plasmids which were amplified in Stable competent E. coli (C3040H, NEB), which were cultivated at 30°C, 150 rpm. Plasmid purification was done in MaxiPrep or GigaPrep scale, using either a manual kit (12162, 12191; QIAGEN) or with an automated method using the PhyPrep system (Biotage AB). Plasmid concentration was quantified using Qubit lx dsDNA BR Assay (Q33266, Invitrogen), purity controlled with NanoDroplOOO (Thermo Scientific) and supercoiling verified on 1% TAE-agarose gel electrophoresis and imaging using Gel Doc EZ System (BIO-RAD). Cargo plasmid ITR integrity were controlled with restriction digestion using Srfl, PvuII-HF and AhdI (NEB), followed by gel electrophoresis. All agarose gel analysis was performed using Image Lab (BIO-RAD).

[0291] Production of AAVs

[0292] Tissue culture-treated 10 cm dishes were seeded with 6 million cells in 10 mL media in the afternoon the day before transfection. The morning after, each plate was transfected with 24 pg of plasmid DNA for AAV2, dHSPG AAV2 and AAV8 or 30 pg of plasmid DNA for Z-AAV variants. The plasmids were transfected in a molar ratio of 1: 1: 1(: 1) Helper:RepCap:Cargo(:Z-VP2). Firstly, plasmids were diluted to 500 pl with OptiPro SFM (10569520, Gibco). In a separate tube, 1 pg / pl PEImax (Polysciences), pH 7.4 in MilliQ water, was added. 48 pg was used for controls and 60 pg for Z-AAV variants. PEI was diluted to 500 pl with OptiPro SFM 84935PC01

[0293] 37 whereby the DNA-OptiPRO mixture was added. The 1 ml was inverted 5 times and incubated for 10 min before dropwise adding the solution to each plate while swirling the plate.

[0294] After 72 hours of incubation, the AAV-containing cell media was aspirated and centrifuged for 10 min at 3260 x g, saving the supernatant. The cells were washed with 2 ml PBS and detached by pipetting with 10 ml PBS-MK (PBS pH 7.4 + 2 mM MgCh, KCI), followed by centrifugation at 500 x g for 5 min. The cell pellet was re-suspended in 1 ml cell lysis buffer (150 mM NaCI, 50 mM Tris-HCI, 2 mM MgC , pH 8.5) per 10 cm plate. Intracellular AAVs were harvested by freezethaw, switching the suspended cells between -80°C dry ice ethanol bath and 37°C water bath in 10-minute intervals, vortexing after each thaw. Lysates were then clarified by 15 min centrifugation at 3260 x g.

[0295] Purification on anti-AAVX resin - AKTA start

[0296] Ahead of purification, cell lysates were treated with 50 U of DENARASE (20804- 100k, c-Lecta) per milliliter of cell lysate for 1 h at 37 °C. Each sample was thereafter pooled with the AAV-containing cell media (lysate and supernatant from two plates were pooled in this step) and filtered through 0.45 pm filter into VivaSpin 20 MWCO 100000 (28932363, Cytiva). The pool was concentrated to 4 ml and then diluted to 10 ml with PBS pH 7.4 + 2mM MgC . The AAVs were purified using 1 ml anti-AAVX columns (A36652, Thermo Scientific) connected to an AKTA start (GE Healthcare, now Cytiva). The column was equilibrated with PBS, pH 7.4, then the prepared AAV sample from 2 plates were loaded, followed by washing with 10 CV PBS before eluting the AAVs with 0.1 M Citric acid, pH 2.5 in fractions of 1 ml for 10 CV. The AAVs were eluted into autoclaved Low proteinbinding tubes (90410, Thermo Scientific). Each fraction was immediately pH- neutralized using 1 M Tris-HCI, pH 8.7, confirming correct pH using pH strips and sterile filtered using 0.2 pm filters. The column was re-equilibrated with 5 CV PBS, whereafter cleaning-in-place (CIP) was performed using 10 CV 0.1 M Citric acid, pH 2.1, followed by 10 CV PBS. A second CIP was performed with 5 CV of 6 M GUA followed by 10 CV PBS, before loading the next sample. Column was stored long-term at 4 °C in 20% EtOH in PBS pH 7.4. AKTA-purification were used for AAV2, dHSPG AAV2, AAV8 and HER2 Z-AAV variants.

[0297] Purification on anti-AAVX resin - MEA2

[0298] The buffers and sample preparation were the same for MEA2 purification as for the AKTA start purifications although the pool of supernatant and cell lysate of 84935PC01

[0299] 38 one 10 cm tissue-culture plate was concentrated to 2 ml and then diluted to 4 ml. AAV-purification was performed using 160 pl anti-AAVX PhyTips on the MEA2 (Biotage AB). All steps were done with a flowrate of 0.5 ml / min. AAVs from the supernatant and cell lysate of one 10 cm plate were captured on two PhyTips. The tips were equilibrated with 1 ml PBS in 4 cycles. Sample capture was done with 2 ml of sample split in two wells, 8 cycles per well. Washing was done with 2 ml of PBS split in two wells, 2 cycles per well. AAVs were eluted using 480 pl elution buffer with 4 cycles. Thereafter immediately pH-neutralized with neutralization buffer and sterile filtered (0.2 pm) into autoclaved Low protein-binding tubes (90410, Thermo Scientific). MEA2-purification was used for all VR4 and VR8 Z- AAV2 variants (except HERZ), HSV-TK AAVs as well as BiZ-AAVs and bivalent controls

[0300] Streptavidin-coated plates (15125, Thermo Scientific) were washed three times with TBS-BT (0.1 % BSA, 0.05 % Tween20), then coated with biotin-conjugated AAV-binding Nanobody AAVX (7103522100, Thermo Scientific) diluted 1 :2000 in TBS-B (0.1% BSA) for 1 hour (h). The plates were washed three times, then AAV samples were added. Standards of AAV2 and AAV8 (RS-AAV2-FL and RS-AAV8-FL, CharlesRiver) were added in serial dilutions of 1 :2 using TBS-B, starting at concentration of around 1011capsids / ml. Samples were diluted 1 : 10 and 1 : 100 dilutions in TBS-B. Plate washed three times and HRP-conjugated AAVX (7303522100, Thermo Scientific) 1 : 10000 dilution in TBS-B was added to each well for 1 h. Plate washed three times and signal detected using TMB (34021, Thermo Scientific), stopping the reaction using 2 M sulphuric acid. Absorbance was measured using Clariostar Microplate Reader (BMG Labtech), standard curve generated with 4PL-adapted curve using MyAssays and analysed using Microsoft Excel. Standard and samples were run in duplicates or triplicates. Viral genome quantification by qPCR

[0301] Purified AAV samples were incubated with DNAse I (New England Biolabs) in a 1 :2 dilution in 2x DNAse I-buffer (20 pl reaction, 0.1 U / pl) for 1 h at 37°C, then heated to 95°C for 10 minutes to heat inactivate DNAse I and to lyse capsids. Samples were diluted serially 1 : 10 and 1 : 100 in DNAse / RNAse free water (10977035, Invitrogen). Cargo plasmid was serially diluted from 2*108to 2*102molecules / pl to use as standard. 2 pl of standard and samples were mixed with a Master Mix consisting of 5 pl iQ™ SYBR® Green Supermix (1708882, BIO-RAD) or 84935PC01

[0302] 39

[0303] SsoAdvanced™ Universal Inhibitor-Tolerant SYBR® Green Supermix (1725018, BIO-RAD), 2.5 pl water, 0.25 pl of 10 pM ITR forward primer and 0.25 pl of 10 pM ITR reverse primer (SEQ ID NO. : 53 and 54) per reaction on 96-well PCR plates (HSP9601, BIO-RAD). qPCR was run in CFX Real-time PCR system (BIO-RAD) using the following programme: 95°C for 2 min, then 40 cycles of 95°C 5 s, 60°C 30 s. A melt curve was generated by increasing the temperature from 65 °C to 95°C in 0.5°C increments every 5 s. Standard curve was created using CFX Maestro Software (BIO-RAD) and data was analysed using Microsoft Excel. All standards and samples were run in triplicates.

[0304] Western blotting

[0305] Purified AAVs were mixed with 3x reducing TCEP-buffer and boiled for 10 minutes at 95°C. The sample was then loaded onto 18 well Criterion gels (5678094, BIORAD) along with PageRuler Plus Prestained ladder (26620, Thermo Scientific). Gel bath was loaded with lx TGS (1610772, BIO-RAD) and run for 40 min at 240V, 4°C. Proteins were transferred to PVDF membranes (1704157, BIO¬

[0306] RAD) using the Trans-Blot Turbo Transfer system (BIO-RAD) on pre-set "Mixed MW" programme.

[0307] The membrane was blocked in 5 % w / v milk powder (Semper) in TBS-T (0.05% Tween20), then stained at 4°C overnight with anti-VPl / VP2 / VP3 antibody (690058, PROGEN) 1 :250 dilution in TBS-T + milk. Subsequently, the membrane was washed three times in TBS-T for 5 min, then stained with secondary antibody anti-mouse HRP-conjugate (31430, Invitrogen) 1:20000 dilution in TBS-T + milk for 1 h. Membranes were washed four times for 5 min, and developed in HRP substrate (WBKLS0500, Merck) before imaging in Chemidoc XRS+ (Bio-Rad) and quantifying VP levels using Image Lab (Bio-Rad).

[0308] Cryo-EM sample preparation, data acquisition and analysis

[0309] Amicon Ultra-2 Centrifugal Filter Units, 100 kDa cut-off (UFC210024, Merck) were pre-soaked with PBS pH 7.4 + 2 mM MgCh, KCI; whereafter purified AAV samples were added and concentrated to > 1012capsids / ml. The concentrate was transferred to Low protein-binding tubes (90410, Thermo Scientific) and stored at -80 °C before process handling for Cryo-EM. From the concentrate, 3 pl were applied to glow-discharged 400 mesh copper grids coated with pure carbon film (TedPella, Inc). The grids were blotted for 3 seconds (s) at 100% humidity and plunge-frozen in liquid ethane using a vitrification robot (Leica EM GP2, Leica Microsystems). The frozen grids were clipped and inserted in a Glacios electron 84935PC01

[0310] 40 microscope (Thermo Fisher Scientific) operated at 200 kV and equipped with a BM-Ceta camera. All cryo-EM images were recorded using EPU software (Thermo Fisher Scientific) with a nominal magnification of 57,000x corresponding to a calibrated pixel size of 5.1 A. The set defocus value for all imaging sessions was - 7 pm for the consistency of the imaging condition. Images were automatically transferred to VAS (QuTEM's proprietary image analysis software) and thirty representative images of each dataset were manually selected based on the vitreous ice quality and thickness. Automated particle detection, verification, refinement, and classification were performed in VAS.

[0311] Each particle in every acquired image was extracted and the particles were compared with the Gromov-Wasserstein distance computed by comparing all particles to a subset of particles in a sequence of largest distance first until convergence. The distance feature was then used as coordinates in a Euclidean space. The median data point representing the median particle was extracted from each sample. Further, the distribution, using random sampling of particle expressions in each sample was compared using Wasserstein distance. The distance matrix was then treated as a Euclidean distance matrix and the corresponding coordinates for each sample were used to compare the morphological difference between the samples.

[0312] Results

[0313] Affibody molecules (abbreviated Z) were integrated into different positions on the AAV, creating Z-AAVs, to find where the affibody molecule would be the most accessible.

[0314] Based on previous peptide fusions in AAVs, the N-terminal of VP2 and two protruding loops of the AAV capsid were chosen as integration sites for affibody molecules. The N-terminal position was denoted N', and the loop integration sites Variable Region 4 (VR4) and Variable Region 8 (VR8) in accordance with previous classifications. The affibody molecule was surrounded by serine-glycine linkers to avoid misfolding of VP2. AlphaFold was then used to predict how the affibody-VP2 (Z-VP2) construct would fold (Data not shown). Because the affibody molecules are attached only to VP2, the Z-VP2 construct had to be expressed separately from the AAV capsid gene (Figure 1A). The first affibody molecule integrated was a HER2-binding affibody molecule (ZHER?), which was grafted onto serotypes AAV2 and AAV8. To accurately study the effect of affibody molecule integration on infectivity, the natural tropism of AAV2 towards heparin-sulphate proteoglycans 84935PC01

[0315] 41

[0316] (HSPGs) was removed. For AAV8, the affibody molecules were grafted to the capsid without prior capsid modification, as AAV8 generally has low infectivity and no strong binding motif has yet been identified.

[0317] The Z-AAVs were produced in HEK293T cells and purified using anti-AAVX columns, where similar elution profiles were seen for all variants (Data not shown). The number of purified viruses were similar or higher for the N' Z-AAV variants compared to wild-type variants (Figure 2A-B). VR4 and VR8 variants produced similarly to each other and around half as efficiently as the wildtype in terms of viral genomes, with lower packaging. Z-VP2 could be detected in all Z- AAV samples except for VR8 Z-AAV8 where the Z-VP2 seems to overlap with VP1 in the Western blot (Figure 2C). This is also clear from the quantification of the Western blots (Figure 2D-E). In general, a ratio towards higher VP1 content is seen in the Z-AAVs, with a slightly lower ratio of Z-VP2 compared to VP2 of the control samples AAV2, dHSPG AAV2 and AAV8.

[0318] Next, the AAVs were visualised by Cryo-EM. To analyse any morphological changes between AAVs in the Cryo-EM images, the Gromov-Wasserstein distances was calculated between all AAVs. The Gromov-Wasserstein distance was used to measure morphological differences between capsid geometries and is unaffected by the rotation or translation of a capsid. By relativising every AAV capsid to all capsids in the same sample as well as to all capsids in related samples, e.g. comparing all AAV2 variants, differences in the capsid structures of AAVs could be analysed without the need for crystallisation. After computing the Gromov- Wasserstein distances, median particles were extracted to provide a representative picture of a packed capsid in each sample (Data not shown). The distribution of morphological variation was then compared by the Wasserstein distance. Distance 0 means that two distributions are identical, i.e., contains the same types of capsid morphologies. The robustness of the analysis was assessed by random sampling of the distributions. Through these comparisons, the inventors found that Z-AAVs with affibody molecules on VR4 or VR8 generally clustered together, indicating similar morphologies, and away from the other samples, indicating diverging morphologies (Figure 3A-B). The unique morphologies of Z-AAVs with affibody molecules on VR4 or VR8 may allow packing of a larger gene into the recombinant AAV. 84935PC01

[0319] 42

[0320] Interestingly, N' Z-AAV8 clustered separately from AAV8 while N' Z-AAV2 clustered together with dHSPG AAV2, indicating higher similarity between these two AAV2 samples than between the two AAV8 samples. AAV2 was seen to cluster separately from dHSPG AAV2 and Z-AAV2-variants.

[0321] Conclusion

[0322] The study shows successful integration of affibody molecules on AAV capsid and the Z-AAVs were evaluated in terms of production levels and morphology of the Z- AAVs. Thus, the study presents a fully modular Z-AAV platform for retargeting AAVs with the help of affibody molecules. This study also demonstrates that the affibody could be integrated on the N-terminal, VR4 or VR8 regions in multiple serotypes exemplified using AAV2 and AAV8. Z-AAV platform has great potential to generate AAVs for selective in vivo targeting.

[0323] Example 2 - Selectivity of Z-AAVs

[0324] Aim of study

[0325] The aim of the study was to assess the selectivity of Z-AAV towards a specific target and their potency.

[0326] Materials and methods

[0327] See Example 1 for the production of Z-AAVs

[0328] Target-ELISA

[0329] High binding 96-well plates (3690, Corning) were coated overnight with biotin- conjugated AAVX (7303522100, Thermo Scientific) in 1: 1000 dilution in PBS or target protein (Table 1) at 10 pg / ml in PBS. The next day, wells were washed twice with PBS-BT (0.1 % BSA, 0.05 % Tween20). PBS-BSA 1 % was added as blocking buffer and incubated for 2 h on slow shake. Wells were then washed once with PBS-BT, and AAV samples diluted in PBS-B (0.1 % BSA) to 1010AAV capsids / ml were added. Samples were incubated for 1 h, then plates were washed three times. HRP-conjugated AAVX (7303522100, Thermo Scientific) 1:5000 dilution in PBS-B was incubated for 1 h. Plate washed three times with PBS, and signal detected using TMB (34021, Thermo Scientific), stopping the reaction using 2 M sulphuric acid. Absorbance was measured using Clariostar Microplate Reader (BMG Labtech) and data normalized to anti-AAVX signal using Microsoft Excel. Samples were run in duplicates or triplicates. 84935PC01

[0330] 43

[0331] Table 1 : Article numbers of proteins used in Target-ELISA

[0332] Transduction assays

[0333] Surface display on ExpiCHO cells was done according to the protocol described in Thalen et al. (Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping. Commun Biol 7, 805 (2024)). Extracellular domains of target proteins were downloaded from UniProt, ordered as gBIocks (IDT) flanked by Ascl / Notl sites and inserted into a GPI-anchor-HA-tag-vector using restriction cloning. Plasmids were purified using MiniPrep (12125, QIAGEN), whereafter concentration and purity were determined via NanoDroplOOO (Thermo Fisher Scientific). ExpiCHO cells were transfected according to the protocol described in Thalen et al. (Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping. Commun Biol 7, 805 (2024)). After 24 hours, expression levels and proper surface folding of the constructs was confirmed by flow cytometry using Cetuximab (EGFR), Trastuzumab (HER.2), Seribantumab (HER.3), a CD40-PDGFRb human bispecific, a VEGFR2 dimeric affibody molecule (ZvEGFR2_i6matG-40matB-PRoss6) or an anti-IGFIR APC-conjugate antibody (17-8849-42, Invitrogen). The human antibodies were detected using an Alexa anti-human 488 antibody (A21445, Invitrogen) and the VEG FR2 -Affibody using a Goat anti-Affibody (Batch no: 0906-PB009, Affibody AB) with an Alexa anti-goat 647 antibody (abl50143, Abeam). Transfection efficiency was determined by anti-HA-tag staining (HA88-342, Invitrogen) for all constructs, detected using Alexa anti-rabbit 488 (A32731, Invitrogen). After confirming expression of constructs, the transfected cells were subcultured to 0.1 MVC / ml, 2 84935PC01

[0334] 44 ml per well in a 24 well plate. 0.5 ml of ExpiCHO Expression Media and purified AAVs (20000 vg / cell for AAV2-variants and 100000 vg / cell for AAV8-variants) were pre-mixed and added to the well. 24 hours post addition, 1.7 ml of cell suspension was spun for 5 min at 300 x g and cells re-suspended in 200 pl PBS-B (1 % BSA). The cells (20 000 cells per sample) were then analysed for GFP-signal in Gallios Flow cytometer or CytoFlex (Beckman Coulter). Data was analysed in Kaluza (Beckman Coulter).

[0335] For the cancer cell transduction assays, 70 000 cells were seeded in 24 well tissue-culture plates. 6 hours later, media was exchanged for media with AAVs at a concentration of 20000 vg / cell for AAV2-variants and 25000 vg / cell for AAV8- variants.. About 42 hours later, the cells were washed, trypsinated and resuspended in media. Thereafter, cells were centrifugated at 300 x g for 5 min and washed twice with PBS-B. Cells were then analysed for GFP-signal in CytoFlex (Beckman Coulter). All samples were run in duplicates.

[0336] Results

[0337] The Z-AAVs were evaluated with respect to their selectivity towards their target HERZ. Binding of the Z-AAVs towards HER2 was evaluated using ELISA by capturing either intact capsids or AAVs bound to HER2. The results showed all VR4 and VR8 Z-AAVs could bind HER2, while the controls did not (Figure 4A). Serotype differences in affibody molecule integration were seen here, as binding was detected between N' Z-AAV8 and HER2 in the ELISA (Fig. 3A, Bottom panel) while binding could not be detected for N' Z-AAV2 (Fig. 3A, Top panel).

[0338] Similar trends were observed when assaying selective cell infectivity. By using a system to display extracellular protein domains on the surface of ExpiCHO-cells. The Z-AAVs were analyzed to evaluate whether it could selectively infect ExpiCHO-cells displaying HER2 (Figure 4B). VR4 and VR8 Z-AAV2 selectively infected the HER2-displaying ExpiCHO-cells, while they did not infect the wild-type cells nor the cells displaying a control construct (RBD of SarS-CoV-2).

[0339] The N' Z-AAV2 barely infected any cell nor did dHSPG AAV2, while AAV2 infected all cells similarly. In the morphological comparisons from Cryo-EM, N' Z-AAV2 clustered together with dHSPG AAV2, while N' Z-AAV8 clustered separately from AAV8 (Figure 3B). This could indicate that the affibody molecule was constricted to the inside of the capsid for N' Z-AAV2, while the larger difference in 84935PC01

[0340] 45 morphology between AAV8 and N' Z-AAV8 potentially could indicate that the affibody molecule is being presented on the outside of the capsid. Large stuffer sequences have previously been reported as key for effective display on the N' of AAV2, further supporting the theory of the affibody-construct being locked inside the capsid in N' Z-AAV2 constructs.

[0341] The Z-AAV8 variants could all infect HER2-GPI, with VR4 and VR8 variants being the most effective, but some off-target infectivity was seen on both wild-type (WT) and RBD-GPI cells for all Z-AAV8 variants. AAV8 could barely infect any cells. To assess if the surface display results translated to human cell lines, all AAV variants were incubated with tumour cells overexpressing HER2 or a control fibroblast cell line (Figure 4C). In line with the surface display results, the VR4 and VR8 Z-AAV2 variants selectively transduced the cells with elevated HER2 expression while showing no infectivity towards the fibroblast cell line.

[0342] All Z-AAV8 variants could infect the cancer cell lines, with VR4 and VR8 being the most effective, and no transduction was seen of the fibroblast control cell line for any AAV8 variant. dHSPG AAV2 and N' Z-AAV2 showed no infectivity to any cell line while AAV2 infected all cell lines.

[0343] Encouraged by these results, the modularity of the Z-AAV platform was tested. As the VR8 Z-AAV2 variant showed the greatest selectivity of HER2-binding (compared to RBD) out of all the Z-AAVs and had higher degree of packaging than Z-AAV8 variants, the VR8 Z-AAV2 was chosen to further test the platform. The results show that integration of at least one cell-type specific binding moiety on VR8 provides a more specific recombinant AAV capsid compared to integration on VR4. Further, integration of at least one cell-type specific binding moiety on VR8 provides a more potent recombinant AAV capsid compared to integration on the N-terminus. Data show a higher level of integration in VR8 than in VR4 for certain variants (Figure 2C).

[0344] Conclusion

[0345] All VR4 and VR8 Z-AAVs could bind HER2.

[0346] In cells, VR4 and VR8 Z-AAV2 selectively infected the HER2-displaying ExpiCHO- cells, while they did not infect the wild-type cells nor the cells displaying a control construct. 84935PC01

[0347] 46

[0348] In a transduction assay, VR4 and VR8 Z-AAV2 variants selectively transduced the cells with elevated HER2 expression while showing no infectivity towards the fibroblast cell line. Z-AAV8 variants infected their target in the Surface display experiment, thus Z-AAV8 variants only infected the cancer cell lines and not the fibroblast control cell lines. This study demonstrates that the addition of a celltype specific binding moiety, such as an affibody, to VR4 or VR8 is serotypeindependent, whereas the addition of said moiety to the N-terminal is serotype dependent, since the cell-type specific binding moiety is only expressed on the surface of AAV8 and not AAV2.

[0349] Example 3 - Retargeting of Z-AAVs

[0350] Aim of study

[0351] The aim of the study was to exchange affibody molecules with new target specificities: EGFR, HER3, IGF1R, PDGFRb or VEGFR2.

[0352] Materials and methods

[0353] See Example 1 for the production of Z-AAVs

[0354] See Example 2 for targeted ELISA and transduction assay

[0355] Results

[0356] Running ExpiCHO surface display with all of the target receptors, selective infectivity was seen for all variants towards their target protein; however, the VR8 ZIGFIR-AAV2 variant did not infect IGFIR-expressing cells (Figure 4D). Correct folding of IGF1R was confirmed using a commercial antibody and extensive testing was then performed to elucidate if VR8 ZIGFIR-AAV2 could infect IGFIR-displaying cells. ZIGFIR-AAVS were incubated with IGFIR-expressing cells without media to remove any potential blocking substrate from the media, the expression of IGF1R was titrated down on the surface of ExpiCHO to not overcrowd the cell surface, and expression system was switched from ExpiCHO to HEK293F to rule out differences in glycosylation; however, none of these tests made the VR8 ZIGFIR- AAV2 infect cells expressing IGF1R (data not shown).

[0357] When testing the infectivity of all the VR8 Z-AAV2s variants on cancer cells, infectivity was indeed seen for the VR8 ZIGFIR-AAV2 against MCF-7 cells, which have elevated IGF1R expression (Figure 4E). VR8 ZHER3-AAV2 could infect SK-BR- 3 and MCF-7 with highest infectivity for SK-BR-3 and VR8 ZEGFR-AAV2 could infect NCI-N87 and SK-BR-3. The cancer cell lines do not express the surface receptors PDGFRP or VEGFR2 and the VR8 ZpDGFRb-AAV2 and VR8 ZVEGFR2-AAV2 did not infect any cancer cell line. No VR8 Z-AAV2 could infect the negative control fibroblast 84935PC01

[0358] 47 cell line. It is presumed that the hDFn cells have low PDGFRb expression, hence no VR8 ZpDGFRb-AAV2 were able to infect the hDFn cells.

[0359] Thus, it is possible to modularly exchange the affibody molecule and direct Z- AAVs towards any target that the affibody molecule binds. Considering the specificity of the viruses in cancer cell infection, a potential therapeutic use was tested of the Z-AAV platform.

[0360] Depending on the use of the Z-AAV platform, higher specificity might be more desirable where limitation of off-target effects is key, while higher potency might be desired if the Z-AAV platform is used to reinforce transduction towards an already established target of the AAV or if off-target effects are less crucial. Additionally, infectivity of all AAV2 and AAV8 variants was higher for SK-BR-3 compared to NCI-N87 and MCF-7 cells. This could possibly be attributed to the upregulated level of the AAV2 and AAV8 co-receptor Laminin Receptor (LamR) in SK-BR-3 compared to NCI-N87 and MCF-7.

[0361] Conclusion

[0362] It was shown that modularly exchanging the affibody molecule and directing the Z-AAVs towards any target is possible.

[0363] 4 - Z-AAVs for use in cancer treatment

[0364] Aim of study

[0365] The aim of the study was to perform a proof-of-concept for the use of Z-AAVs in cancer treatment.

[0366] Materials and methods

[0367] See Example 1 for the production of Z-AAVs

[0368] Proof-of-concept assay using HSV-TK and ganciclovir (GCV).

[0369] In 96-well tissue-culture plates, 5000 cells (NCI-N87) or 3000 cells (SK-BR-3 and hDFn) were seeded in 100 pl of media. Adjacent wells were filled with 200 pl of sterile water to reduce evaporation. Plates were wrapped with plastic foil, then incubated for 6 hours at 37 °C, 5% CO2 for cells to adhere whereafter the media was exchanged for media containing AAVs at a concentration of 100000 vg / cell. The plates were re-wrapped and incubated for 48 hours with the AAVs. The AAV- media was then removed, and media containing ganciclovir (GCV) (SML2346-1ML, SigmaAldrich) at concentrations of 0 pM, 10 pM and 100 pM were added. After 93 hours of incubation, 10 pl of alamarBlue HS (A50100, Invitrogen) was added and incubated for 3 hours for a total of 96 hours of incubation with ganciclovir. Plates 84935PC01

[0370] 48 were then analysed in Clariostar Microplate Reader (BMG Labtech) with recommended settings for detection of resorufin (Ext: 545 - 20, Emi: 600 - 40) and adjusting gain to 80% of the wells where neither AAVs nor ganciclovir was added to cells. All cell, AAV and ganciclovir combinations were run in 6 replicates over two 96-well plates. Two-way ANOVA statistical analysis comparing no GCV samples to 10 pM or 100 pM samples was performed using Prism (GraphPad).

[0371] Results

[0372] As a proof-of-concept for the use of Z-AAVs in cancer treatment, the inventors utilized the prodrug system of herpes simplex virus thymidine kinase (HSV-TK) in conjugation with ganciclovir (GCV). The HSV-TK enzyme converts ganciclovir into a dGTP mimic, inhibiting replication and has previously been reported for other AAV cancer prodrug assays.

[0373] VR8 Z-AAV2S targeting HER2, PDGFRb and VEGFR2 along with AAV2 and dHSPG AAV2, all of them carrying the gene for HSV-TK, were produced. VR8 ZpDGFRb-AAV2 and VR8 ZVEGFR2-AAV2 functioned as negative controls for VR8 ZHER2-AAV2. The viruses were purified using the MEA2 purification protocol and then evaluated based on the capacity to induce cytotoxicity in NCI-N87 and SK-BR-3 cells, which have the highest elevated expression of HER2 out of the cancer cell lines assayed (Figure 4E). The fibroblast hDFn cell line was used as negative control.

[0374] Highly significant declines in NCI-N87 proliferation was seen only for AAV2 and VR8 ZHER2-AAV2 at both low (10 pM) and high (100 pM) concentrations of ganciclovir (GCV) (Figure 5A). A less significant decline was observed for VR8 ZpDGFRb-AAV2 at 100 pM ganciclovir. For SK-BR-3, significant declines in proliferation were again only seen for AAV2 and VR8 ZHER2-AAV2 (Figure 5B). Lastly, for hDFn, only AAV2 resulted in a significant decline in proliferation (Figure 5C). For both SK-BR-3 and hDFn, some variants show significant increases in proliferation at increasing ganciclovir concentrations, such as VR8 ZpDGFRb-AAV2.

[0375] Conclusion

[0376] With these results, a potential therapeutic application was demonstrated where Z- AAVs specifically target and hinder tumour cell proliferation. This demonstrates a potential use of the platform for specific targeting in cancer treatment. The present example also demonstrates that the Z-AAV system of the present 84935PC01

[0377] 49 invention is more specific than wildtype AAV2 and would therefore result in less off-target effects that AAV2.

[0378] Aim of study

[0379] The aim of this study was to use the Z-AAV platform to generate dual receptortargeting AAVs.

[0380] Materials and methods

[0381] See Example 1 for the production of Z-AAVs

[0382] Results

[0383] As phenotype switching between surface receptors can be an issue in tumour targeting, the Z-AAV platform was used to generate dual receptor-targeting AAVs.

[0384] When a tumour cell is treated by targeting a specific receptor, such as blocking HER.2 with trastuzumab, the cancer sometimes switches phenotype and upregulates other receptors to compensate. These compensatory receptors can be e.g. HER.3, IGF1R or others. To combat phenotype switching affecting the infectivity of Z-AAVs, targeting two different cell receptors at the same time by bispecific Z-AAVs (BiZ-AAVs) was explored. These carry two affibody molecules targeting different receptors: HER.2 and HER3, HER2 and IGF1R, or HER3 and IGF1R. For example, the bispecific Z-AAV targeting HER2 and HER3 may have the sequence as presented in SEQ ID NO. 29-32.

[0385] The affibody molecules were attached to both VR4 and VR8 on VP2 of dHSPG AAV2 (Figure IB). Each combination of ZHER2, ZHERS, and ZIGFIR fused to both VR4 and VR8 was tested, generating a total of six different BiZ-AAV variants and three bivalent Z-AAV controls with the same affibody molecule on both VR4 and VR8 as controls. Titres were comparable for all BiZ-AAVs to the single VR4 Z-AAV2 and VR8 Z-AAV2 variants (data not shown). Western blotting showed the presence of BiZ-VP2 for all variants (data not shown).

[0386] ELISA showed BiZ-AAVs could bind both of their targets (Figure 6A) while the bivalent controls and single Z-AAV2s could only bind their specific target (Figure 6B). To test whether the BiZ-AAVs were still potent, the viruses were incubated with MCF-7 cells, which has been shown to switch phenotype when treated against HER2. Results showed that the BiZ-AAVs could transduce cells, although overall infectivity was lower than for the single VR8 ZHER2-AAV2 variants (Figure 6C). The BiZ-AAVs were more effective at infecting MCF-7 than the bivalent Z- 84935PC01

[0387] 50

[0388] AAVs, indicating bivalency only had detrimental effects on AAV infectivity. The most effective BiZ-AAV had ZHERS on VR4 and ZHER2 on VR8, where infectivity increased compared to the single ZHER3-AAV2 variants, although still lower than single ZHER2-AAV2 variants. Additionally, ZIGFIR seemed to only have a detrimental effect on infectivity for the BiZ-AAVs compared to having a single ZIGFIR on VP2. The relatively high potency of the ZHER2-VR4: ZHER3-VR8 BiZ-AAV indicates that viruses would still be infectious if the MCF-7 cells were to phenotype switch to upregulate HER3 instead of HER2.

[0389] Conclusion

[0390] Bispecific BiZ-AAVs showed the ability to bind two target proteins. This effect is particularly useful if the tumour cell switches phenotype as a compensatory mechanism to avoid detection by a target protein. The AAV capsid could also comprise more than two cell-type specific binding moieties.

[0391] Example 6 - Affibodv-VP2 constructs with reduced or without linkers Aim of study

[0392] The aim of the study was to investigate the effectivity of Z-AAV with reduced or without linkers.

[0393] Materials and methods

[0394] See Example 1 for the production of Z-AAVs

[0395] Results

[0396] The linker used in this study comprises serine and glycine, in particular S4G linkers according to SEQ ID NO. : 59. Different linker lengths between VR8 and the ZHER2-Affibody molecule were tested on VR8 Z-AAV8. Starting from the original construct (4L-Z-5L), linkers (L) were reduced on different sides of the affibody molecule in varying degrees (3L-Z-3L, 1L-Z-1L, 3L-Z-1L, 3L-Z-0L, 1L-Z-3L, 0L-Z- 3L) all the way down to a no linker control (OL-Z-OL) (Figure 7A). The constructs were evaluated in regard to capsid integrity, production levels, packaging ability and, most importantly, selectivity and potent infectivity.

[0397] Surprisingly, the construct without linkers was the best performing variant out of all Z-AAVs in all categories. It showed one of the strongest binding signals to HER2 in an ELISA-setup (Figure 7B), as well as having the highest and most selective infectivity towards HER2-displaying ExpiCHO-cells (Figure 7C).

[0398] Additionally, the linker-free Z-AAV produced very well, similarly to wild-type AAV8 and with similar packaging degree (Figure 7D). 84935PC01

[0399] 51

[0400] Conclusion

[0401] The construct without linkers was a well-performing variant. Surprisingly, these results indicate that no linkers could result in an increased target binding and increased selectivity. It is therefore beneficial to avoid using linkers when grafting the ZHER2-Affibody to VR8 of Z-AAV8.

[0402] Example 7 - Functionality of linker-free BiZ-AAVs and linker scouting Aim

[0403] The aim of the study was to investigate if linker-free BiZ-AAVs could be generated and which linker length was most potent.

[0404] Materials and Methods

[0405] See example 1 for the production of Z-AAVs.

[0406] Results

[0407] Affibody molecules (SEQ ID 33-34, 37-38 or 39-40) were grafted without linkers to VR4 and / or VR8 of AAV2 (SEQ ID 11-12, SEQ ID 13-14). A schematic representation of linker-free grafting to both VR4 and VR8 are shown (Figure 8A)

[0408] In the target-ELISA, all new variants bound their target protein only. Similar binding signals was seen between almost all monovalent variants with or without linkers (Figure 8B). Binding signal was similar for the BiZ-AAVs variants with or without linkers (Figure 8C). When assessing selective infectivity towards HER2 and HER3 using surface display on ExpiCHO, the bispecific ZHER3 / ZHER2 noL BiZ- AAV had the highest infectivity of all Z-AAVs towards both the HER2-displaying and the HER3-displaying cells (Figure 8D).

[0409] All new noL Z-AAVs and BiZ-AAVs were also tested on MCF-7 cells along with their full linker counterparts. As shown earlier (Figure 6C), MCF-7 cells were less transduced when using full linker BiZ-AAVs compared to the monovalent variants. However, the data from the linker-free Z-AAVs show that the most potent out of all tested Z-AAVs was the ZIGF1R / ZHER2 noL BiZ-AAV2 (Figure 8E).

[0410] Conclusion

[0411] The BiZ-AAVs without linkers, in most applications demonstrated, were the most potent in infectivity, improving upon the previous full-linker BiZ-AAVs. 84935PC01

[0412] 52

[0413] Example 8 - In vivo mice study of linker-free Affibody-AAV

[0414] Aim

[0415] The aim of the study was to explore if Affibody-AAVs could be retargeted to other tissues in a mouse ( / n vivo).

[0416] Materials and methods

[0417] See example 1 for the production and purification of Z-AAVs. For the AAV samples used in the in vivo study, elution in the AKTAstart system was done with 0.1 M Citric acid, pH 2.0. The purest AAV-containing fraction (3rd fraction) were used for the in vivo study after dialysis in PBS using Slide-A-Lyzer cassettes (66300, Thermo Scientific), with subsequent filtering (0.1 pm).

[0418] In vivo study

[0419] Mice (Male and female C57BI / 6J BomTAC between 3-4 months old and weighing 23.1-36.2 g) were randomly divided into four groups: n = 6 / group (4m / 2f) for the AAV samples and n = 4(2m / 2f) for PBS control. A bolus dose of 5E+1010viral genomes in 100 pL PBS was injected via tail vein. Control animals received the equivalent volume of PBS. Weight was monitored twice per week after injection. Mice were euthanized 14 days post injection and transcardially perfused with 0.9% NaCI. Organs (brain (left hemisphere), liver, heart, lung, spleen, pancreas, kidneys, muscle (left hindlimb)) were collected and snap-frozen in dry ice.

[0420] Frozen tissues were homogenized in lOOul TBS per 40mg tissue in 2 ml tubes packed with 1.4 mm ceramic beads (Precellys P00912-LYSK0A). All organs underwent the "medium soft tissue" programme (3x 10s, 6500 rpm; with 15s pause between cycles) on Precellys 24 Touch. Heart tissue underwent the programme twice. After centrifugation at 3 min, 21000g, supernatants (soluble cytosolic fraction) were kept for quantification of GFP. Samples were kept at -80 °C and thawed on ice prior to use. eGFP in supernatants was quantified using ELISA (abl71581, Abeam) and the previously mentioned Clariostar. Samples were diluted at least 1:5 in the kit's buffer per manufacturer's recommendation. Samples or standard dilutions were run in duplicates. Standards ranged from 4.7 pg / ml to 2400 pg / ml. A linear curve was fitted to standards from 4.7 pg / ml to 1200 pg / ml, and an exponential curve 84935PC01

[0421] 53 to standards from 150 pg / ml to 2400 pg / ml. Samples were preferably quantified using linear curve. Samples above the linear standard curve were quantified using the exponential curve. Samples below the standard was set to 0 pg / ml. Analysis was done in Microsoft Excel and statistical analysis was done using Prism. For Figure 9C-D, ordinary one-way ANOVA was performed followed by Tukey's multiple comparisons test with a single pooled variance, to compare the mean of each column for a given tissue. For Figure 9E, ordinary two-way ANOVAs were performed followed by Sidak's multiple comparisons test with a single pooled variance, comparing the mean of each sub-column (male / female or >28g / <28g) to its neighbouring sub-column.

[0422] Results

[0423] The following AAVs were injected into the mice: wild-type AAV2 (SEQ ID 1-2), VR8 noL ZIGF1R-AAV2 (SEQ ID 13-14, ID 39-40) or VR8 noL ZPDGFRb-AAV2 (SEQ ID 13-14, ID 41-42), packing an eGFP gene as DNA cargo (Figure 9A). The Z-AAVs were well-tolerated by the mice as seen from the small weight differences before, during and after treatment (Figure 9B). Analysing eGFP- expression in tissue homogenates, the Z-AAVs were re-directed towards other tissues than the liver (Figure 9C-D). The PDGFRb-targeted Z-AAV was found primarily in the heart, kidney and muscle tissue, while the IGFIR-targetted Z-AAV was found in several tissues, such as muscle, lung and heart. Notably, only the IGFIR-targeted Z-AAV showed significantly high eGFP-expression in the brain and pancreas. AAV2 expression was primarily found in the liver, with very large difference in expression if separating the mice based on gender or weight (Figure 9E, 9F). A significant increase of eGFP expression in liver was seen in male mice compared to female mice, but this correlation was also statistically significant when comparing to initial weight, where the female mice were under the average weight of 28 grams and the male mice above it (Figure 9E, 9F). A similar correlation is seen for lung and muscle, where eGFP expression was higher in males and by weight for the IGFIR-targeted or PDGFRb-targeted Z-AAV2. No other tissues showed statistically significant differences comparing gender or weight.

[0424] Conclusion

[0425] Affibody-AAVs are effectively de-targeted from the liver and into other organs. 84935PC01

[0426] 54

[0427] Sequence listing 84935PC01

[0428] 55 84935PC01

[0429] 56

Claims

84935PC0157Claims1. A recombinant adeno-associated virus (AAV) capsid comprising an engineered virion protein (VP), wherein the engineered VP comprises at least one cell-type specific binding moiety located in variable region 8 (VR8), wherein the cell-type specific binding moiety comprises a three-helix bundle and a length selected from the range of 40 to 150 amino acids.

2. The recombinant AAV capsid according to claim 1, wherein the length of the cell-type specific binding moiety is in the range of 45 to 140 amino acids, such as in the range of 50 to 130 amino acids, such as in the range of 55 to 120 amino acids, such as in the range of 55 to 110 amino acids, such as in the range of 55 to 100 amino acids, preferably in the range of 40 to 90 amino acids, more preferably in the range of 45 to 80 amino acids, most preferably in the range of 50 to 80 amino acids.

3. The recombinant AAV capsid according to any one of the preceding claims, wherein the three-helix bundle is a three alpha-helix bundle.

4. The recombinant AAV capsid according to any one of the preceding claims, wherein the recombinant AAV capsid further comprises at least one additional celltype specific binding moiety.

5. The recombinant AAV capsid according to claim 4, wherein the additional celltype specific binding moiety is located on a protruding structure of the recombinant AAV capsid, such as a protruding loop, an alpha-helix, or a betasheet.

6. The recombinant AAV capsid according to any one of claims 4 or 5, wherein the protruding structure is located at a position selected from the group consisting of N-terminus, variable region 1 (VR1), variable region 4 (VR4), variable region 5 (VR5), variable region 6 (VR6), between VR5 and VR6, variable region 7 (VR7), and variable region 8 (VR8), preferably VR4.84935PC01587. The recombinant AAV capsid according to any one of claims 4-6, wherein the at least one additional cell-type specific binding moiety is identical to or different from the cell-type specific binding moiety located in VR8.

8. The recombinant AAV capsid according to any one of the preceding claims, wherein the cell-type specific binding moiety is specific for one or more surface receptors expressed on a target cell, such as a mammalian cell.

9. The recombinant AAV capsid according to any one of the preceding claims, wherein the cell-type specific binding moiety is selected from the group consisting of Affibody molecule, ABD-Derived Affinity Protein (ADAPT), de novo designed proteins, such as a3D, TIP-98, TIP-99, GB-CCW9, GBB-CW11, and BAAB-CCW8, or a combination thereof.

10. The recombinant AAV capsid according to any one of the preceding claims, wherein the cell-type specific binding moiety is an Affibody molecule.

11. The recombinant AAV capsid according to any one of claims 9 or 10, wherein the Affibody molecule is selected from the group consisting of SEQ ID NO. 33 to 44, and a combination thereof.

12. The recombinant AAV capsid according to any one of the preceding claims, wherein the cell-type specific binding moiety is flanked by a linker or is not flanked by a linker.

13. The recombinant AAV capsid according to any one of the preceding claims, wherein the engineered VP is selected from the group consisting of an engineered virion protein 1 (VP1), engineered virion protein 2 (VP2), and engineered virion protein 3 (VP3), preferably engineered VP1 or engineered VP2, more preferably engineered VP2.

14. The recombinant AAV capsid according to any one of the preceding claims, wherein the AAV is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAVDJ, AAVrhlO, AAVrh74, AAV-PHP, AAV-PHP.eB, AAV-LK03, AAV2.5, AAV5.2,84935PC0159AAV-Anc80, and AAV-SparklOO, preferably AAV2, AAV5, AAV8 and AAV9, more preferably AAV2 or AAV8.

15. A recombinant adeno-associated virus (AAV) particle comprising the recombinant AAV capsid according to any one of the preceding claims.

16. A genetic construct comprising a nucleotide sequence encoding a recombinant AAV capsid according to any one of 1-14.

17. A system for delivering at least one nucleic acid to a cell or tissue determined by a cell-type specific binding moiety, said system comprises a recombinant adeno-associated virus (AAV) particle according to claim 15.

18. The recombinant AAV particle according to claim 15 or the system according to claim 17 for use as a medicament.

19. The recombinant AAV particle according to claim 15 or the system according to claim 17 for use in the treatment, alleviation, and / or prevention of a disease selected from the group consisting of cancer, Alzheimer's disease, Parkinson's disease, epilepsy, Autosomal dominant polycystic kidney disease (ADPKD), Nephronophthisis (NPHP), lipoprotein lipase deficiency, Leber's congenital amaurosis, Spinal muscle atrophy (SMA), Haemophilia A, Haemophilia B, Duchenne muscular dystrophy (DMD), and Aromatic L-amino acid decarboxylase (AADC) deficiency, preferably cancer.

20. A method for producing the recombinant AAV particle according to claim 15, said method comprising the steps: al) providing a first genetic construct comprising a nucleotide sequence encoding an engineered virion protein (VP) comprising a cell-type specific binding moiety located in variable region 8 (VR8), or a2) providing a first genetic construct encoding an adeno-associated virus (AAV), wherein the start codon of a virion protein (VP) is mutated, and providing a second genetic construct comprising a nucleotide sequence encoding an engineered VP comprising at least84935PC0160 one cell-type specific binding moiety located in variable region 8 (VR8), b) transfecting the first genetic construct of step al) or the first genetic construct and the second genetic construct of step a2) into host cells in conditions suitable for said host cells to produce AAV particles, and c) isolating AAV particles, wherein the mutated VP and the engineered VP are the same VP variant.

21. The method according to 20, wherein the genetic construct is selected from the group consisting of messenger RIMA (mRNA), doggybone DNA (dbDNA), plasmid, yeast artificial chromosome (YAC), and bacterial artificial chromosome (BAC).

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