Synthesis and application of magnetic particle conjugates for the extracorporeal removal of antibodies targeting viral vectors to facilitate gene therapy

Abstract

The present invention provides novel conjugates comprising a magnetic particle and a multitude of viral structures covalently bound to said particle. These conjugates are suited for efficient extracorporeal removal of antibodies from blood. In particular, the conjugates are suited as adjuvant treatment in gene therapy.

Description

[0001] Synthesis and application of magnetic particle conjugates for the extracorporeal removal of antibodies targeting viral vectors to facilitate gene therapy

[0002] The present invention provides novel conjugates comprising a magnetic particle and a multitude of viral structures covalently bound to said particle. These conjugates are suited for efficient extracorporeal removal of antibodies from blood. In particular, the conjugates are suited as adjuvant treatment in gene therapy .

[0003] It is well known that gene therapies based on genetically engineered adeno-associated viruses (AAV) or adenoviruses (AdV) are a therapeutic option to successfully treat monogenetic diseases (Wang et al., Nature Review Drug Discovery, 2019, doi : 10.1038 / s41573-019-0012-9 and Mendell et al., Mol. Ther. Methods Clin. Dev., 2022, 26;25:74-83) . However, the influence of pre-existing immunity to AAV, or AdV respectively, negatively effects - or even prevents - gene therapy, particularly when genetically engineered viruses are administered intravenously. Both neutralizing and non-neutralizing antibodies have an impact on the efficacy and safety of gene therapies based on viral vectors. While neutralizing antibodies directly prevent vector transduction, both neutralizing and non-neutralizing antibodies when bound to a viral vector have the potential for complement activation leading to immune-mediated toxicities. Additionally, vectors can trigger, or boost in case of pre-existing immunity, the adaptive immune response consequently leading to an increase in the levels of anti-vector antibodies making the re-dosing of the viral vector difficult. Re-dosing is often necessary to achieve the intended therapeutic efficacy.

[0004] Bertin et al (WO2018 / 158397 ) describe a method for removing undesired anti-AAV neutralizing antibodies from a blood-derived composition. The method described in that document comprises contacting blood derived composition with at least one support onto which one or more affinity ligand (s) specific for anti-AAV antibodies is bound. Although suitable, the method described in that document is considered disadvantageous, as the removal of anti-AAV antibodies is insufficient and / or treatment times are long. Without being bound by theory, it is believed that restricted movement and comparatively low accessible surface area of the support with affinity ligands are limiting factors.

[0005] Boedecker-Lips et al (J Clin Apher. 2023; 38: 590-601) disclose a study to understand the efficiency and timing of anti-AAV antibody removal by immunoadsorption with regenerative broadband columns. The document concludes in that such regenerative broadband columns represent a potentially safe and effective strategy to increase the patient population amenable to AAV-based gene therapy by lowering pre-existing anti-AAV antibodies to below threshold levels. Although suitable, the method described in that document is considered disadvantageous, as the chosen approach is non-specific meaning that it removes a broad range of antibodies, the removal of anti-AAV antibodies is insufficient and / or treatment times are long .

[0006] Yun et al. (J Clin Neurosci. 2012; 19: 875-880) describe adenoviral vectors labeled with superparamagnetic iron oxide nanoparticles comprising bi-functional carboxylpolyethylene glycol ) 8-amine (CA[PEG]8, for use as a contrast agent to increase MRI specificity. According to this document, a multitude of nanoparticles are linked to an adenoviral capsid, thereby yielding viral capsids labeled with nanoparticles.

[0007] Orlowski et al. (Mol. ther. Methods clin. dev. 2020, 16: 192-203) describe removal of anti-AAV antibodies from blood via hemapheresis using an immunoadsorbent matrix comprising Sepharose beads coupled to AAV9 virions. According to this method, anti-AAV antibodies are removed by passing the blood through an immunosorbent column comprising the sepharose beads and a filter preventing the passage of said sepharose beads , while allowing passage of blood cells .

[0008] In consequence , there is a need for further, particularly improved methods to remove antibodies from blood, thereby allowing or improving gene therapy .

[0009] Thus , it is an obj ect of the present invention to mitigate at least some of these drawbacks of the state of the art . In particular, it is an aim of the present invention to provide improved methods for blood puri fication and improved therapeutic approaches towards gene therapy .

[0010] These obj ectives are achieved by providing conj ugates as defined in claim 1 , formulations as defined in claim 13 , methods for removing antibodies from blood as defined in claim 18 , and methods for administering a viral vector as defined in claim 21 . Further aspects of the invention are disclosed in the speci fication and preferred embodiments are disclosed in the speci fication and the dependent claims .

[0011] The present invention will be described in more detail below . It is understood that the various embodiments , preferences and ranges as provided / disclosed in this speci fication may be combined at will . Further, depending on the speci fic embodiment , selected definitions , embodiments or ranges may not apply .

[0012] Unless otherwise stated, the following definitions shall apply in this speci fication :

[0013] As used herein, the term " a" , " an" , " the" and similar terms used in the context of the present invention ( especially in the context of the claims ) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context .

[0014] As used herein, the terms "including", "containing" and "comprising" are used herein in their open, non-limiting sense .

[0015] As used herein, the term "gene therapy" refers to the treatment, prevention, or delay of progression of a disease by introducing genetic material into cells. In certain embodiments, the introduced genetic material replaces and / or complements defective or missing genes, and / or alters the genetic code of genes, and / or modifies gene expression. In certain other embodiments, the introduced genetic material encodes a therapeutic protein, such as an antibody. Particularly, the term includes methods of gene editing in a cell, in particular gene editing using CRISPR-Cas based systems, such as CRISPR-Cas9, e.g., SaCas9 and SpCas9, as well as Casl2a, Casl2f, Casl3d, Cas-Phi, and Casl2b. CRISPR-Cas based systems also include prime editing systems and base editors. In the context of the present application, the cells are part of a living organism in need of such treatment, particularly a human being (i.e. a "patient") . In the context of the present application, it is to be understood that the cell is a somatic cell (i.e. a cell of a mammalian body, including stem cells, excluding germline cells) . Accordingly, human germline cells and human germline stem cells are excluded when referring to cells .

[0016] As used herein, a "gene" refers to a DNA region (including exons and introns) encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions. In the context of gene therapy, the genetic material introduced into a cell could be a part of a "gene" (such as the cDNA) or entirely recombinantly synthesized (such as DNA encoding a single-chain variable fragment of an antibody, or codon-optimized cDNA etc.) .

[0017] As used herein, a "patient" is a human being suffering from or diagnosed with a disease, in particular a disease susceptible to, treatable by, or manageable by gene therapy. At the time of performing a method provided herein on a patient, the patient may or may not exhibit symptoms of the disease.

[0018] As used herein, the term "viral structures" includes (i) viral capsids (VCs) with recombinant genetic material e.g., DNA or RNA, (ii) VCs without recombinant genetic material, (iii) individual viral capsid proteins, and (iv) assemblies of viral capsid proteins. Such viral structures comprise at least one epitope capable of being bound by an antibody against the viral vector of interest (such as a specific serotype of AAV) . Thus, in certain embodiments, a viral structure can be a fragment of a viral capsid, wherein the fragment comprises such an epitope. Fusion proteins where heterologous amino acid sequences are fused to the amino acid sequence of such an epitope can also be used with the methods and compositions described herein.

[0019] As is known in the field, the term "viral capsid" refers to the protein shell of a virus surrounding the virus' nucleic acid genome. In nature, viral capsids enclose the virus' genetic material. Such viral capsids comprising genetic materials are also referred to as "nucleocapsids " when they form a discrete substructure within a virus, e.g., in enveloped viruses. In the context of the present invention, the presence of genetic material inside the viral capsid is not required. Nevertheless , it is also possible that genetic material is present inside the viral capsid . The term "viral structures" also includes nucleocapsids .

[0020] The proteins making up the viral capsid are commonly called "viral capsid proteins" , also referred to as viral coat proteins . As is known in the field, viral capsid proteins are typically oligomeric proteins , which typically comprise several repeating, oligomeric structural units . The individual protein subunits that assemble to form said oligomeric structural units are also referred to as protomers . Thus , the term "viral structures" , and in particular the term " individual viral capsid proteins" , also includes "protomers" and assemblies of protomers , provided said protomers or said assemblies thereof are capable of being bound by antibodies .

[0021] The term "oligomeric protein" is not to be confused with the term "oligopeptide" . As is common in the field, the term "oligomeric protein" refers to a protein complex comprising several structural protein units , which may either be repeating, identical units ( also referred to as "homo-oligomeric" ) or di f ferent units ( also referred to as "hetero-oligomeric" ) . "Viral capsid proteins" typically comprise identical , repeating oligomeric structural units .

[0022] In contrast , the term "oligopeptide" refers to individual amino acid chains comprising several amino acids , typically up to a length of 15 amino acids .

[0023] In the context of this invention, VCs may or may not be enclosed by a viral envelope , i . e . , a protecting membrane consisting of lipids and proteins that protect the viral capsid . In the context of this invention, viral capsid proteins are understood as proteins that have the function to maintain the shape and structural integrity of a virus .

[0024] As used herein, the term "blood" shall include whole blood, blood plasma, and blood serum . As used herein, the term "antibody" (sometimes abbreviated as "Ab") shall include total antibodies present in the blood, both neutralizing and nonneutralizing antibodies. It thus typically refers to full-length immunoglobulins, excluding fragments. However, to the extent binding fragments of antibodies interfere with a gene therapy of interest, the methods and compositions provided herein are similarly suited to reduce the titer of such binding fragments of antibodies.

[0025] The present invention will be better understood by reference to the figures .

[0026] Figure 1. A schematic representation of the synthesis of conjugates comprising particles with VCs according to example 1. From left to right:

[0027] 1) Core-type particles with cores having soft magnetic properties; said Particle covered by a first shell containing graphene layer.

[0028] 2) Said particle covered by a second shell of polyglycerol, NHS activated.

[0029] 3) Conjugate, comprising viral capsids covalently bond.

[0030] Figure 2. Illustrates examples 2.2 and 2.3: Depletion of fluid (buffer, plasma, blood) from anti-viral capsid antibodies by incubation with conjugates and subsequent magnetic separation. From left to right

[0031] • Left: Inventive conjugate + blood containing anti-AAV antibodies and / or anti-AdV antibodies

[0032] • Right: purified blood (depleted in anti-AAV antibodies and / or anti-AdV antibodies) and antibody-conjugate- complex separated by applying a magnetic field.

[0033] Figure 3: Quantification of rAAV9 (rAAV9 with genetic material) and rAAV90(rAAV9 without genetic material) VCs bound to particles and antibody removal by conjugates in PBS . • The percentage of rAAV9 VCs (black column) and rAAV90VCs (white column) bound to particles was quantified by measuring the VCs in the supernatant before and after coupling (A) .

[0034] • The antibody removal by conjugates with rAAV9 VCs (black column) or rAAV90VCs (white column) in ADK9- spiked (1 pg / mL) PBS was determined by measuring the amount of ADK9 before and after incubation with conjugates (B) .

[0035] The graphs represent n=2 conjugate batches, which were each measured in duplicate (A) or triplicate (B) . The mean ± SD is shown.

[0036] Figure 4. Conjugate treated human whole blood and plasma. Conjugate treatment (2 mg / mL, 1 treatment cycle, 50 minutes) was performed in whole blood and plasma generated from the respective whole blood.

[0037] The antibody levels before (left) and after treatment with inventive conjugate (right) were measured in an ELISA-based assay in triplicates (y-axis: fluorescence, a.u.] . The dotted horizontal line represents the average value of the low titer donors.

[0038] Figure 5. Performance of conjugates coupled to capsid comprising genetic material ("filled capsid") versus empty capsids (i.e., capsid without genetic material) . The removal of anti-AAV9 antibodies in human plasma after conjugate treatment, with rAAV9 or rAAV90, treatment was measured for 3 donors. The plasma was treated for 3 cycles of 3 minutes and measured in either an ELISA-based assay [ (A) , antibody removal, fluorescence (a.u.) ] or in a virus neutralization assay [ (B) , transduction efficiency, (%) ] . The results from the 1:3 dilutions are depicted. The dotted horizontal line represents the average value of the low titer donors. The mean and SD are depicted.

[0039] From left to right: untreated - treated with rAAV9 conjugate - treated with rAAV90conjugate. Figure 6: illustrates the inventive method for removing anti-AAV and / or anti-AdV antibodies from blood in an ex vivo setting.

[0040] Figure 7: illustrates a Gene therapy applying the inventive method to deplete anti AAV / AdV Ab first, conducting the gene therapy afterwards; followed by optional repeating these steps. Y-Axis: AAV / AdV titer; X- Axis: time (not scaled)

[0041] In more general terms, in a first aspect, the invention relates to new conjugates comprising magnetic particles as defined herein which are covalently bound to a multitude of viral structures as defined herein. This aspect of the invention shall be explained in further detail below:

[0042] In an advantageous embodiment, the invention relates to a Conjugate comprising a particle and a multitude of viral structures. In certain embodiments, a multitude of viral structures is a plurality of the same viral structure (i.e., multiple copies of the same viral structure) . In certain other embodiments, the multitude comprises multiple different viral structures, wherein each such viral structure can be present as a plurality of such viral structure (ie, multiple copies of the same viral structure) . In certain embodiments, provided herein are such Conjugates wherein (a) said particle has a size of 5 nm to 5 pm and is of the core-shell type and comprises: (i) a core, said core consists or consists essentially of an iron - based material having soft magnetic properties; covered by a (ii) first shell, said shell contains one or more graphene layers which are optionally functionalized; and / or covered by (iii) a second shell, said second shell contains a polymer selected from the group consisting of Polyglycerol, dextran, Hydroxyethyl starch, Polylactic acid (PLA) , Styrene-maleic acid copolymers (SMA) , Polystyrene (PS) , Poly ( ethylene imine) , Chitosan, Polymethyl-methacrylate (PMMA) ; with the proviso that, if no second shell is present, said one or more graphene layers are functionalized; with the proviso that the particle shells do not contain magnetic material; and (b) said viral structures are based on genetically engineered adeno-viruses (rAdV) or genetically engineered adeno- associated-viruses (rAAV) ; and are selected from viral capsids (VCs) with recombinant genetic material, VCs without recombinant genetic material, individual viral capsid proteins, and assemblies of viral capsid proteins; and (c) said viral structures are covalently bound to said particle shell.

[0043] In an advantageous embodiment, the viral structures are viral capsids, such as VCs with genetic material (e.g. recombinant genetic material) and VCs without genetic material, e.g., VCs without recombinant genetic material.

[0044] Typically, between 2 and 2000 of the viral structures are bound to the particle, e.g., 2 - 100 of the viral structures (e.g. viral capsids, with or without genetic material) , such as 2 - 50 of the viral structures. In certain embodiments, between 2 and 50 of the viral structures are bound to the particle.

[0045] In certain embodiments, the ratio of particle : viral structures (e.g. viral capsids, with or without genetic material) is between 1 : 2 and 1 : 2000, e.g., between 1:2 and 1:100, such as between 1:2 and 1:50.

[0046] Thus, in certain embodiments, the particles have a loading of between 2 - 2000 viral structures (e.g. viral capsids, with or without genetic material) per particle, such as a loading of between 2 - 100, e.g. between 2 - 50, viral structures per particle.

[0047] The number of viral structures bound to the particle or the ratio between particle and viral structures can be determined using standard methods. For example, it can be determined by ELISA, e.g., as described in example 1. In certain embodiments, provided herein is a conjugate comprising a particle and a viral structure, wherein the particle comprises: a) a core with soft magnetic properties with a coercive force below 30 000 A / m and a magnetic saturation above 30 emu / g; and b) a shell surrounding the core, wherein the shell does not comprise magnetic material and wherein the shell is suitable to reduce degradation of the core; c) prior to conjugate formation, a functional group capable of covalently bonding to the viral structure; and wherein the viral structure is covalently bound to the particle via the functional group. In more specific embodiments, the viral structure of such a conjugate comprises an epitope of a viral capsid of AAVrh74 or AAV9.

[0048] In certain embodiments, the particle comprises both the first shell and the second shell. This embodiment is preferred when the core comprises 10 wt% - 100 wt% iron carbide. The amount of iron carbide may be determined using X-ray-diffraction (XRD) .

[0049] Magnetic Particles

[0050] In certain embodiments, the particles provided herein are suitable for use with the devices described in international patent application publications

[0051] WO 2020 / 058136 and WO 2021 / 063708. As the skilled artisan will appreciate in view of the present disclosure, physical and magnetic properties of the Conjugates provided herein can be adjusted to the specific requirements of the device being used.

[0052] Particle Size:

[0053] Particle size may be determined by electron microscopy, dynamic light scattering (DLS) or analytical centrifugation. When using DLS or analytical centrifugation, the hydrodynamic diameter is measured. Particle size may vary over a broad range, e.g., from 5 nm to 5 gm, preferably 5 nm to 0.6 gm. This range covers the particle size as measured by dynamic light scattering as well as by electron microscopy and includes nanoparticles of 5 nm up to and including 100 nm and microparticles of more than 0.1 gm up to and including 5 gm.

[0054] In certain embodiments, the particle size is between 20 nm - 5 gm, such as between 20 nm - 150 nm, e.g., between 40 nm - 100 nm, as determined by electron microscopy.

[0055] In certain embodiments, the particle has a hydrodynamic diameter of 5 nm - 600 nm, e.g., 100 nm - 300 nm, as determined by Dynamic Light Scattering (DLS) . In certain embodiments, particle size can be between 5 nm to 200 nm if measured using DLS. In certain embodiments, the D(90) value and / or the D(50) value for the particle size can be between 5 nm to 200 nm or between 5 nm and 100 nm if measured using DLS.

[0056] In certain embodiments, the conjugate has a hydrodynamic diameter of 5 nm - 600 nm, e.g., 100 nm - 600 nm, such as 100 nm - 300 nm, such as 150 nm - 250 nm, e.g., 160 nm - 220 nm, as determined by DLS.

[0057] In certain embodiments, the conjugate has a hydrodynamic diameter of 20 nm - 300 nm, e.g., 35 nm - 200 nm, such as 50 nm - 100 nm as determined by analytical centrifugation .

[0058] Particle structure:

[0059] Particles according to this invention are of the core shell type, i.e. an entity of a first material forming a core and one or more second materials fully covering said core and thereby forming one or more shells. This allows a number of different types of particles, referred to as type 1 ... type 4 particles:

[0060] • Type 1 particles contain, particularly consist of, a core as described herein which is covered by said first shell, which contains one or more graphene layers that are functionalized. In a specific embodiment, multiple graphene layers constitute said first shell, wherein the outermost graphene layer is functionalized or wherein only the outermost graphene layer is functionalized.

[0061] • Type 2 particles contain, particularly consist of, a core as described herein which is covered by said second shell, said second shell contains or consist of a polymer as defined herein.

[0062] • Type 3 particles contain, particularly consist of, a core as described herein which is covered by said first shell, which contains one or more graphene layers that are not functionalized; and which is covered by said second shell, said second shell contains or consist of a polymer as defined herein.

[0063] • Type 4 particles contain, particularly consist of, a core as described herein which is covered by said first shell, which contains one or more graphene layers that are functionalized; and which is covered by said second shell, said second shell contains or consist of a polymer as defined herein. In a specific embodiment, multiple graphene layers constitute said first shell, wherein the outermost graphene layer is functionalized or wherein only the outermost graphene layer is functionalized.

[0064] Particle Core:

[0065] A wide range of iron-based materials may be used, preferably iron-based material which contains at least 50 wt% Fe . This includes iron alloys, iron oxides and iron carbides .

[0066] In certain embodiments, the core comprises 10 wt% - 100 wt% iron carbide, e.g., 20 wt% - 100 wt%, such as 50 wt% - 100 wt%. Iron carbide includes e.g., FeaC, FeaC, FeaCa . In certain embodiments, the core comprises 5 wt% - 90 wt% elemental iron (Fe(0) ) , e.g., 10 wt% - 40 wt%, such as 10 wt% - 30 wt%.

[0067] In certain embodiments, the core comprises a total content of iron carbide and elemental iron of between 90 wt% - 100wt% of the core mass, e.g., 95 wt% - 100 wt%, such as 98 wt% - 100 wt%.

[0068] In certain embodiments, the core comprises less than 5 wt% of iron oxide (i.e., 0 - 4.9 wt% iron oxide) , e.g., less than 1 wt% of iron oxide (i.e., 0 - 0.9 wt% iron oxide) .

[0069] The composition of the core can be analysed using known techniques, in particular using XRD.

[0070] Materials suitable in the context of this invention show soft magnetic properties, preferably with a coercive force below 30'000 A / m and a magnetic saturation above 30 emu / g. Preferably, the coercive force is below 16'000 A / m. In an ideal case, the coercive force is zero, resulting in a superparamagnetic material. Magnetic properties can be adjusted depending on the device being used. Such a device can be a device as described in international patent application publications

[0071] WO 2020 / 058136 and WO 2021 / 063708. For example, magnetic properties can be chosen to prevent binding of the particles to each other. Without being bound by theory, magnetic properties that would result in binding of the particles to each other could result in clogging of the device being used. In certain embodiments, the system of Conjugates provided herein and a device as described in international patent application publications

[0072] WO 2020 / 058136 and WO 2021 / 063708 allows for reduction of the titer of a specific antibody by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% in at most 120 min wherein the titer is determined by an antibody binding assay.

[0073] The core size may vary. Typically, the core has a size of 5 - 150 nm, preferably 10 - 100 nm, e.g., 10 nm -50 nm, such as 20 nm - 50 nm, as measured by electron microscopy. It is preferred to have a narrow and uni- modal size distribution. Typically, more than 90 %, preferably more than 95 % (n / n) are within a defined range . As outlined above, the particles are of the core-shell type, containing one particle core. However, due to manufacturing, agglomeration of particle cores may take place, thus forming a matrix-type core with a size up to 150 nm. Such agglomerated particles typically account for less than 20 % (n / n) , preferably less than 10% of particles .

[0074] Particle shell (s) : According to this invention, neither the first shell nor the second shell does contain magnetic material. It is considered beneficial to provide particles where the magnetic material is protected, not exposed to the environment (particularly blood) , and not exposed to viral structures. In certain embodiments, a particle shell increases stability of the particle. As such, a shell protects the core from oxidative degradation. In a specific embodiment, a shell for use with the compositions provided herein provides protection of the core from oxidation which would result in an observed weight gain, which can be measured by gravimetry. Additionally, the core is protected from acidic or basic media such as HC1, which would result in the dissolution of the core, which can be detected by gravimetry. In certain embodiments, a particle shell protects the core's stability by 50%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of degradation compared to the same core without shell .

[0075] First Shell:

[0076] The first shell is optional. Suitably, the first shell comprises preferably of 3 - 15 Graphene layers. Choosing the term "graphene" indicates that the carbon atoms are predominantly (or almost exclusively) present in the sp2- hybridization state without additional atoms bound. In a specific embodiment, only those carbon atoms that are functionalized have an additional bond to a functional group such as a functional group of Formula (I' ) (see below) . Due to its size, such shells are sometimes also characterized as "Super-Buckminster-fullerenes" . Further advantageous embodiments of said shell are explained below .

[0077] Preferably, the graphene layer has a thickness between 0.3 and 10 nm, particular preferably 1 - 5 nm (as evaluated from transmission electron micrographs, corresponds to ~ 3 — 15 graphene layers) . This results in a carbon content (as measured by quantitative microanalysis using a LEGO-900) of between 0.5 and 20 %wt . Such shells are dense and thus prevent oxidative degradation of the particle core. It is thus preferred to implement graphene layers as a first shell where the material of the particle core is sensitive towards oxidation, such as iron, iron alloys and iron carbides. In embodiments, the graphene layer is free of functional groups. This embodiment is implemented in case a second shell is present. In a specific embodiment, all graphene layers of a first shell are free of functionalization. In embodiments, the graphene layer contains functional groups on its outermost layer. Such functional groups, also termed linkers, improve adhesion of the second shell or allow covalent bonding to the viral structures. Thus, such functional group (I ) serves as a linker and spacer. Suitable linkers are of formula (I ) wherein the squiggly line represents a graphene layer, n is an integer between 1 and 5,

[0078] FG represents independent from each other OR, NHR, NR2,

[0079] COR, COOR, SR, SOR, SO2R, P(O)OR, and P(O)2OR,

[0080] R represents independent from each other H, C1-C4 alkyl, C1-C4 alkoxy, C3-C10 cyclo-alkyl, and C4-C10 aryl.

[0081] In preferred embodiments, n represents 1 and FG represents OH or OR.

[0082] It is understood that the functional group FG will react with either the viral structure or the polymer of the second shell, forming a covalent bond. The structures shown herein are prior to such reaction.

[0083] In embodiments, the first layer has a thickness between 1 - 20 nm, such as 2 - 10 nm. For example, a particle consisting of a core having a size of 10 - 50 nm and the first layer, but without the second layer, typically has a size of 12 - 60 nm, as measured by electron microscopy.

[0084] Second Shell:

[0085] If present, a broad range of polymers is suited as a second shell. In view of the intended use in pharmacy, suitable polymers are preferably compatible with blood. Further, said polymer contains functional groups allowing, directly or after activation, covalent bonding to viral structures. Suitable are polymers based on synthetic monomers, including Polyglycerol, Styrenemaleic acid copolymers (SMA) , Polystyrene (PS) , Poly ( ethylene imine) , and Polymethylmethacrylate (PMMA) . Also suitable are polymers based on naturally occurring monomers, including dextran, Hydroxyethyl starch, Polylactic acid (PLA) , and chitosan.

[0086] In an advantageous embodiment, the polymer is polyglycerol, particularly hyperbranched polyglycerol. In another embodiment, the second shell contains a polymer selected from the group consisting of Polyglycerol, Polyethylene glycol (PEG) , dextran, Hydroxyethyl starch, Polylactic acid (PLA) , Styrenemaleic acid copolymers (SMA) , Polystyrene (PS) , Poly ( ethylene imine) , Chitosan, Polymethylmethacrylate (PMMA) .

[0087] Depending on the type of particle, the polymer of the second shell is either covalently bound to the linker of formula (I ) (type 4 particles) , or without covalent bond to the first shell (type 3 particles) , or in direct contact with the core, with or without bonds, (type 2 particles) .

[0088] If a second shell is present, the viral structures are covalently bound to said second shell. Suitable linkers of said second shell may be selected from thioethers and amides, . In certain embodiments, thioethers and amides have the general formula -SR' ' and -C(O)NHR' ', respectively, where R' ' represents C1-C4 alkyl, C1-C4 alkyloxy, or an amino acid. In certain embodiments, such a linker of said second shell is covalently bound to a polymer of said second shell, and in turn is suitable to conjugate to a viral structure. In embodiments, the polymer accounts for 10 wt% - 60 wt% of the particle including second shell (but not counting the viral structure of a Conjugate) .

[0089] In a preferred embodiment, the particle comprises a first shell comprising a linker of formula (I' ) discussed above; a second shell consisting of or consisting essentially of a hyperbranched polyglycerol which is covalently bound to the linker of formula (I' ) on the inside of the shell and which contains linkers selected from thioethers -SR' ' and amides -C(O)NHR' ' where R' ' represents C1-C4 alkyl, C1-C4 alkyloxy, or an amino acid on the outside of the shell, thereby allowing covalent bonding to the viral structures.

[0090] In embodiments, the second shell has a thickness between 30 nm to 50 nm. In certain embodiments, a particle consisting of a core, a first layer and a second layer has a particle size between 40 nm to 110 nm, as measured by electron microscopy.

[0091] Viral Structures

[0092] The term viral structure is defined above. In the context of this invention, viral structures are selected from genetically engineered adeno-viruses (AdV) and genetically engineered adeno-associated-viruses (AAV) . Specifically, recombinant AAV particles, capsids, or epitope-comprising fragments thereof can be used with the compositions and methods provided herein. Illustrative serotypes of AAV from which viral structures can be obtained and produced recombinantly to generate Conjugates are: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrhlO, AAVrh8, AAV-rh74, and hybrid serotypes of the foregoing. Suitable AAV serotypes further include myotropic AAVs, such as AAVMYO and myoAAV. As the skilled person understands based on the present disclosure, recombinant AAV particles, capsids, or epitope-comprising fragments also include genetically engineered capsids, such as bCapl and eCapl, available from DynoTherapeutics . In certain embodiments, the term "genetically engineered" excludes natural serotypes (wild type) . Further, the term "genetically engineered" is chosen to cover both, recombinant viral structures obtainable by classical genetic engineering methods (recombinant AdV or AAV) and viral structures obtainable by gene editing methods (e.g., Crispr / Cas9) .

[0093] In embodiments of the invention, the viral structure is based on recombinant adeno-viruses (rAdV) , which includes all serotypes of adeno-viruses.

[0094] In embodiments of the invention, the viral structure is based on recombinant adeno-associated-viruses (rAAV) , which includes all serotypes of adeno-viruses.

[0095] In embodiments of the invention, the viral structures are selected from viral capsids and individual viral capsid proteins, preferably viral capsids, in particular viral capsids without genetic material, i.e., viral capsids being essentially free of genetic material.

[0096] In certain embodiments, conjugate comprising a core, a first layer, a second layer and a multitude of viral structures has a size between 65 nm - 160 nm, as measured by electron microscopy.

[0097] Covalent Bonding / Conjugate

[0098] The term "conjugate" is chosen to describe an entity comprising a magnetic particle and one or more viral structures, where said viral structures are covalently bond to said magnetic particle. Such conjugate is schematically shown in fig.l, right side. A multitude of viral structures is covalently bound. The number of viral structures depends on the size of the particle, the number of bonding sites available and the size of the viral structure. It is preferred to have 2 - 2000 viral structures covalently bound to the particles surface, e.g., 10 - 1000 viral structures, such as 2 - 100 viral structures or 2 - 50 viral structures. This may be quantified by measuring the concentration of unbound viral structures that remain in the liquid phase after the coupling reaction (see example 1) . Unbound viral structures may be measured using ELISA.

[0099] One or more covalent bonds may be present to link a viral structure to the particles surface. It is considered beneficial that already one single covalent bond allows a stable and reliable linkage of viral structure to particle surface. Without being bound to theory, it is believed this covalent bonding does not, or almost not, influence the 3D viral structure and thus enables the beneficial applications discussed below.

[0100] In embodiments, the conjugate has a zeta potential < 0 mV.

[0101] In embodiments, to reduce or avoid the formation of particle aggregates due to cross-linking, a blocking agent containing at least one free amine, such as human serum albumin, glycine or TRIS, is conjugated together with the viral structure in ratios of viral structures to blocking agent of e.g. 1:100, 1:10, 1:1, 1:0.1, 1:0.01.

[0102] Formulations

[0103] Suitably, the inventive conjugates are handled, applied or administered in the form of a liquid formulation. Such formulations contain the inventive conjugates and a dispersion medium.

[0104] Suitably, the dispersion medium is selected from pharmaceutically acceptable aqueous media, preferably buffered aqueous media. Such dispersion media are known to the skilled person and include balanced salt solutions, such as PBS buffer. Suitably, the concentration of the inventive conjugates is in the range of 20 - 200 mg / mL.

[0105] Suitably, the formulation has a pH in the range of 5 - 9. Suitably, the osmolality of the formulation is in the range of 200 - 400 mOsm / kg.

[0106] Such liquid formulations comprise a multitude of con ugates .

[0107] In embodiments, the formulation contains one single type of conjugates. In this embodiment, all conjugates contain the same particle and the same viral structure on its surface. This is beneficial if it is known that the patient has antibodies against a specific viral vector (e.g., a serotype of AAV, such as AAV9) .

[0108] In embodiments, the formulation contains two or more types of conjugates (a "cocktail") . In this embodiment, a 1stgroup of conjugates contains a 1stgroup of viral structures, a 2ndgroup of conjugates contains a 2ndgroup of viral structures, and a nthgroup of conjugates contains a nthgroup of viral structures on its surface. This is beneficial since the conjugate cocktail could be adjusted to the viral vectors to be used for the gene therapy if more than one is used (e.g., different serotypes of AAV or AdV) , cross-reactive antibodies that could interfere with the gene therapy could be removed, and finally a cocktail of conjugates could be used as one treatment that fits all intended gene therapies and all patients .

[0109] In embodiments, the formulation as described herein is a sterile formulation.

[0110] In certain embodiments, the ratio of particle : viral structures in the liquid formulation is between 1:2 and 1:2000, e.g., between 1:2 and 1:1000, such as between 1:2 and 1:50. In this embodiment, the ratio of particle : viral structures is calculated based on the total amount of conjugates present in the formulation (i.e., the multitude of conjugates present in the formulation) . The ratio of particle : viral structures may be determined as described above and in the context of example 1. The amount of viral structures can be measured by ELISA.

[0111] In certain embodiments, the size distribution of the conjugates in the liquid formulation is between 60 nm - 300 nm, e.g., between 100 nm - 300 nm, as measured by dynamic light scattering. Advantageously, the polydispersity index (PDI) is between 0.01 - 0.3.

[0112] In a second aspect, the invention relates to new uses / applications of the inventive conjugates described herein. Generally speaking, the inventive conjugates are suited to remove antibodies present in a fluid, specifically blood ("blood purification") and as adjuvant therapy to gene therapy ("gene therapy") . This aspect of the invention shall be explained in further detail below:

[0113] Blood purification

[0114] In embodiments, the invention provides for conjugates as described herein for use in the removal of antibodies from blood, wherein the antibodies being selected from the group consisting of anti-AAV antibodies and anti-AdV antibodies .

[0115] For this purpose, a sample of blood is taken from a patient's circulation and treated before it is returned to the circulation. The therapeutic assembly to perform the described immunoadsorption comprises an extracorporeal flow line, which is defined here as the entire apparatus carrying the blood outside of the body. For this purpose, the extracorporeal flow line comprises two connection ports to the patient, wherein a first connection port comprises the inlet of untreated body fluid from the patient or the inlet of the sample of untreated body fluid of the patient into the flow line. A second connection port comprises the outlet of treated body fluid to the patient from the flow line. In an embodiment, the connection ports are located as to allow a veno-venous flow line. In an alternative embodiment, the connection ports are located to allow a veno-arterial flow line.

[0116] Between the two connection ports, an extracorporeal flow line carries the blood outside of the body, driven by a fluid pump to pump the blood through the extracorporeal flow line. The extracorporeal flow line further contains an injection point for injecting inventive conjugates (step a below) followed by a section allowing binding of antibody and conjugate (step b below) , which in turn is followed by a device for magnetic removal of antibodyconjugate complexes and non-reacted conjugates (step c below) . Such therapeutic assembly is shown in fig. 6. In certain embodiments, a device as described in international patent application publications

[0117] WO 2020 / 058136 and WO 2021 / 063708 can be used with the compositions and methods provided herein.

[0118] In embodiments, the invention provides a method for removing antibodies from blood, the antibodies being selected from the group consisting of anti-AAV antibodies and anti-AdV antibodies, and the method comprising: a. contacting the blood with a conjugate or a formulation as described herein; subsequently b. mixing the thus obtained composition to thereby effect binding of said antibodies to said conjugate to obtain an antibody-conjugate-complex; and subsequently c. separating the antibody-conjugate-complex from the blood by means of a magnetic material.

[0119] The method is schematically shown in fig. 2 and illustrated in fig. 6 in more detail.

[0120] It is considered a substantial benefit of the inventive method that the conjugates contacting the blood are capable of unrestricted movement within the blood to be treated. Further, due to the small size of the conjugates used, they possess a large accessible specific surface area. Without being bound by theory, it is believed that these two attributes synergistically interact and therefore positively enhance mass transfer rates and augment the adsorption capacity of the antibodies relative to conventional methodologies described above. For example, in embodiments, 6 g of the conjugates described herein, e.g., being present in a liquid formulation with a volume of 50 ml, are sufficient for the purification of up to 12 L of blood (i.e., removal of antibodies) .

[0121] It is considered particularly beneficial that such small amounts of conjugates, and consequently such small volumes of liquid formulation, are sufficient for blood purification. For example, such small amounts of conjugates or such low volumes of liquid formulation result in lower dead volumes and thus reduced blood loss of the patient.

[0122] Moreover, conjugates of the present invention selectively bind to target antibodies, and thus exhibit little to no non-specific binding to other blood constituents.

[0123] Finally, due to its magnetic properties, a safe removal of conjugates and complexes is achieved without contaminating the patient. Overall, the method provides improved removal efficiency, reduced treatment times and enhanced safety for the patients benefit when compared to the conventional treatments described above.

[0124] Step a. Contacting may be implemented by an injection step, in which, by means of an injection device, a formulation according to the first aspect is added to the patients' blood present in the extracorporeal flow line. The formulation containing the inventive conjugate is added in a therapeutically effective dose, i.e. comprising the conjugate at a concentration and dose necessary to reduce the antibody titer. Injection may be controlled in terms of at least one of the following: injection rate, time of injection, injection dose, concentration, injection pressure.

[0125] Step b. Mixing of the blood and the conjugate may be affected in a conventional way, e.g. by tubular flow in the extracorporeal flow line for a time sufficient for binding. It is understood that the inventive conjugates are mixed with the patient's blood prior to step c.

[0126] Step c. Separation may be achieved by passing the blood containing antibody-con ugate complex through a magnetic field. Such magnetic field may be formed by a permanent magnet. This step ensures the patients' titer is reduced to a level allowing further therapy, specifically gene therapy as discussed below.

[0127] In embodiments, the antibody is an anti-AAV antibody. In this embodiment, the conjugate comprises viral structures based on genetically engineered adeno-associated-viruses (AAV) . For example, anti-AAV antibodies include, but are not limited to, antibodies against the following AAV serotypes: AAVrh74, AAV9, AAV8, AAV2, AAV5, and myotropic AAVs, such as AAVMYO and myoAAV. In certain embodiments, anti-AAV antibodies are selected from antibodies against AAVrh74 and AAV9.

[0128] In embodiments, the antibody is an anti-AdV antibody. In this embodiment, the conjugate comprises viral structures based on genetically engineered adeno-viruses (AdV) .

[0129] In embodiments, the antibodies comprise both anti-AAV antibodies and anti-AdV antibodies. In this embodiment, either the conjugate comprises viral structures based on genetically engineered AAV and based on genetically engineered AdV; or the conjugate is a cocktail where a first group of conjugates comprises viral structures based on genetically engineered AAV and a second group comprises conjugates based on genetically engineered AdV.

[0130] As used herein, the removal of antibodies includes both, full and partial removal. In the context of an adjuvant therapy to gene therapy, it is not required to fully remove specific antibodies from patient's blood. Rather, a substantive reduction of the titer allows an improvement in gene therapy. Accordingly, in embodiments, the titer of said antibody in the blood, measured within 24 hrs after removal, is reduced to less than 50%, preferably less than 35%, of the initial titer. The titer may be determined using standard methods, such as an antibody binding assay or a neutralizing antibody assay.

[0131] In embodiments, said removal of antibodies involves (i) continuous removal of blood from a patient prior to step (a) , and (ii) continuous returning of blood which is depleted from said antibodies after step c to said patient. Accordingly, the method is, in an advantageous embodiment, an extracorporeal treatment of a patient. It is considered a substantial benefit that the conjugates as described herein are not in direct contact with the human body, thereby mitigating risks and facilitating medical approval processes at the same time. This treatment may take place for a period of 0.5 - 5 hrs. It comes as a surprise that such short extracorporeal treatment times are sufficient to significantly reduce patient's titer in anti-AdV-antibodies and / or anti-AAV antibodies. In certain embodiments, use of a Conjugate provided herein with a device such as a device as described in international patent application publications WO 2020 / 058136 and WO 2021 / 063708 can reduce the titer of antibodies against a gene therapy vector of interest by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% in at most 120 min wherein the titer is determined by an antibody binding assay

[0132] In embodiments, said removal is performed in one to five sessions. Often, one single session is sufficient to allow gene therapy as described below.

[0133] In embodiments, both neutralizing and non-neutralizing antibodies are removed. This embodiment allows safe application of a subsequent gene therapy.

[0134] Gene therapy Viral vector-based Gene therapy involves administering a viral vector to a patient. Accordingly, the invention relates to conjugates and formulations as described herein, for use in AdV- or AAV based Gene therapy.

[0135] According to this invention, said viral vector is selected from genetically engineered AAV vectors and genetically engineered AdV vectors, preferably selected from rAAV and rAdV vectors. The inventive gene therapy involves two steps. First step: removing the subject's circulating antibodies by the method described above ("blood purification") . Second step: administering the viral vector to the patient. Said first and said second step may be repeated for 1 - 10 times. This therapeutic approach is outlined in fig. 7. Accordingly, the inventive gene therapy comprises two steps, said first step as outlined above and said second step, the gene therapy as known in the field. Said known gene therapy comprises

[0136] • The loading of gene-payload into AAV / AdV vectors to obtain said genetically engineered vector, and

[0137] • Injecting said vector to the patient thereby delivering of genes to target cells and expression in said cells to thereby obtain a therapeutic effect. In embodiments, this injection is performed within 1 hour - 7 days after completing the first step.

[0138] In embodiments, the viral vector is a vehicle for Gene therapy. The goal of gene therapies, such as, for example, for monogenetic diseases, is the long-term correction of the disease phenotype in an affected patient. To achieve this, viral vectors may be adapted

[0139] • to introduce genetic material of choice into cells of the patient (e.g., genetic material encoding a therapeutic protein, which can be a protein heterologous to the patient) ; or

[0140] • to replace a disease-causing gene with a healthy copy of that gene; or • to inactivate a disease-causing gene that is not functioning properly; or

[0141] • to introduce a new gene into the body to help treat a disease; or

[0142] • to introduce a modified gene into the body to help treat a disease.

[0143] Accordingly, the invention relates to a Viral vectorbased Gene therapy and a method of administering a viral vector to a patient, where said vector is adapted (i) to replace a gene, (ii) to inactivate a gene, (iii) to introduce a new gene, or (iv) to introduce a modified gene, to thereby treat a disease.

[0144] In certain embodiments, the term "gene therapy" relates to in vivo CRISPR-Cas gene editing. For example, suitable CRISPR-Cas systems include systems based on Cas9, such as SaCas9 and SpCas9, Casl2a, Casl2f, Casl3d, Cas-Phi, SpCas9, and Casl2b. Suitable CRISPR-Cas systems also include prime editing systems and base editors.

[0145] In embodiments, the viral vector is an AAV vector. In embodiments, the viral vector is an AdV vector. Such viral vectors are most commonly used in gene therapy, where AAV based gene therapy represents the majority of current and expected future treatments.

[0146] By lowering the antibody titer in a patient, according to the first step described above, the full potential of gene therapy is unlocked. First, a patient re-dosing is possible, thereby increasing efficacy. Second, an expanded access to patients, previously excluded due to high titers of neutralizing antibodies, is enabled.

[0147] Third, the inventive gene therapy improves transduction efficiency, due to a potentially reduced dosing and thus reduced toxicity concerns. Forth, complement activation is limited which reduces safety issues.

[0148] In certain embodiments, a particular gene therapy may first be administered to a patient who tests negative for antibodies against the specific viral vector used for the gene therapy (eg, negative for the specific serotype of AAV) . In certain embodiments, testing negative means that the patient's antibody titer is below the specified threshold for a particular gene therapy. Re-dosing of the same gene therapy at a later time to the same patient, however, may be neutralized by then-existing antibodies against the viral vector. Methods provided herein allow for re-dosing in such patients by reducing the titer of such neutralizing antibodies in the patient's blood.

[0149] In view of the above, the inventive gene therapy may be applied in the context of treatment with viral vectors commercially available under the trade names Zolgensma ®, Roctavian ®, Elevidys ®, Upstaza ®, Luxturna ®, and Hemgenix ®.

[0150] Further provided herein is a method of reducing the titer of an antibody against a virus in a mammal comprising: a) contacting blood of the mammal with a conjugate provided herein (such as the conjugate described in the preceding paragraph) under conditions suitable for binding of the antibody to the conjugate; and b) magnetically separating the antibody-conjugate- complex from the blood in a magnetic field.

[0151] In specific embodiments, in such a method for reducing the titer of an antibody, the viral structure of the conjugate used with the method comprises an epitope of a viral capsid of AAVrh74 and wherein the antibody is an anti-AAVrh74 VC antibody. In certain more specific embodiments, the mammal is a non-human primate or a pig. In additional, more specific embodiments, the mammal is a human patient; and / or the method is conducted extracorporeally ; and / or the antibody titer is reduced by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% in at most 120 min wherein the titer is determined by an antibody binding assay. Also provided herein is a method of performing a virus- vectored gene therapy on a patient , wherein the method comprises in the following order : a ) administering the gene therapy to the patient ; and b ) performing a method described herein for reducing the titer of an antibody against a viral vector used as a vector in the gene therapy; and c ) re-administering the gene therapy to the patient .

[0152] In a third aspect, the invention relates to a process for manufacturing conj ugates as described herein and liquid formulations as described herein . This aspect of the invention shall be explained in further detail below :

[0153] In embodiments , the invention provides for a process for manufacturing a conj ugate as described herein, 1staspect , the process comprising the steps of : a ) providing a first suspension comprising core-shell particles as described herein; b ) providing a second suspension / dilution comprising viral structures as described herein; c ) combining said first and second suspension to allow reacting the viral structures with the particles to form a covalent bond, optionally in the presence of an auxiliary reagent , optionally by applying heat or ultrasound, to thereby obtain a suspension comprising the conj ugate described herein; d) optionally further treatment of the suspension obtained in step c ) .

[0154] It is considered beneficial that the inventive process is applicable to a wide range of core-shell particles and to a wide range of viral structures by applying coupling chemistry known to the skilled person . The inventive process shall be explained in further detail below, referring to the steps a ) ... d) Step a) : Suspending particles in a dispersing medium is known in the field. To improve reaction in step c) and / or to de-agglomerate particles, the suspension of step a) may be subjected to ultrasonication or vortexing. Suitable diluents include aqueous solutions optionally containing pH adjusting agents, surface-active agents, viscosity modifiers and / or complexing agents. Suitable are, for example, PBS buffer containing EDTA.

[0155] Step b) : Depending on the viral structure and the diluent, the material provided may be in the form of a suspension or in the form of a solution. To reflect this situation, the term 'second suspension / dilution ' is chosen. Suitable diluents are as described above, step a) . Suitable are, for example PBS buffer containing Glycerol and non-ionic surfactants.

[0156] Step c) : Viral structures contain free amine groups on its surface. These groups are capable in forming of a covalent bond with a reactive group present on the particle. Such reactions are known per se in organic chemistry, but not yet applied to the present starting materials (i.e. Viral structures and core-shell particles as described herein) .

[0157] Step d) , if applied, involves one or more of the following steps: dl) separating the obtained conjugates from the suspension, d2 ) lyophilizing the thus obtained conjugate, d3) purifying the thus obtained suspension.

[0158] In embodiments, sterilization takes place after step c) or step d) .

[0159] In embodiments, the invention provides for manufacturing a liquid formulation as described herein, the method comprising the step of e) combining conjugates as described herein (e.g. the composition obtained in step c) or d) or isolated ('dry' or 'lyophilized' ) conjugates) with a diluent compatible with blood, e.g. a pharmaceutically acceptable aqueous solution.

[0160] To further illustrate the invention, the following examples are provided. These examples are provided with no intend to limit the scope of the invention.

[0161] Example 1: Conjugate synthesis

[0162] Example 1.1 Synthesis of core-shell particles with polymer ('type 4 particles' ) conjugated to rAAV9 VCs Example 1.2 Synthesis of core-shell particles with polymer ('type 4 particles) conjugated to rAAV90VCs

[0163] The synthesis of conjugates comprising VCs (1) with and (2) without genetic material is schematically depicted in Figure 1 and explained in detail below.

[0164] Materials & Methods:

[0165] Stocks of viral capsids (VC) of recombinant Adeno- Associated Virus Serotype 9 (rAAV9) with genetic material (rAAV9, "filled capsid") and without genetic material (rAAV90, "empty" capsid) were provided in PBS pH 7.2 + 0.001% Pluronic F68 + 5% glycerol at 3.78E13 vgc / mL rAAV9 (equivalent to 1E14 - 1.3E14 VC / mL) and 3E14 vgc / mL rAAV90(equivalent to 2.24E14 VC / ml) . The VC stocks were diluted to IxlO13VCs / mL in PBS.

[0166] Magnetic graphene-coated iron-carbide core-shell particles coated with a hyperbranched polyglycerol were synthesized as described in [1] , [2] and [3] . The polymer -OH groups were then reacted to -COOH groups via succinylation as described in [4] . The carboxyl groups were then activated using a one-pot reaction with EDC / sulfo-NHS as described in [5] to thereby obtain NHS- activated core-shell particles. The respective chemical reaction methods described in references [1] , [2] , [3] , [4] , and [5] are hereby incorporated by reference.

[0167] The NHS-activated core-shell particles with semi-stable amine-reactive NHS-esters on the surface were washed and resuspended in PBS buffer (containing 1 mM EDTA) at a particle concentration of 2 mg / mL. Vortexing and ultrasonication for 5 min were applied to homogeneously disperse the particles. The NHS-activated particle suspension was then immediately added to the previously prepared solution of rAAV9 VC (or rAAV90VC, respectively) at a particle concentration of 1 mg / mL. After ultrasonication for 1 min, this reaction mixture was then shaken for at least 3 h at 900 rpm at room temperature. The NHS-activated particles react with free amines on the VCs forming a covalent amide bond. The resulting particles-VC conjugates were sonicated for 30 sec and subsequently magnetically separated. A small sample of the supernatant was removed to determine the concentration of unreacted VC (see below section "Quantification of VC bound to particles") . The conjugates were again resuspended using ultrasonication (1 min) and a quenching solution of Tris buffer (20 mM) was added to the suspension. After mixing for 10 minutes at 500 rpm, the conjugates were washed three times with PBS containing 1 mM EDTA and then resuspended in PBS at concentrations of 5 mg / mL.

[0168] Literature :

[0169] [1] R. N. Grass, W. J. Stark, WO 2008 / 055371.

[0170] [2] I. K. Herrmann et al, Synthesis and Covalent Surface Functionalization of Nonoxidic Iron Core-Shell Nanomagnets, Chem. Mater. 2009, 21, 3275-3281.

[0171] [3] S. Doswald et al, Low molecular weight glycerol derived coatings on magnetic nanoparticles, RSC Adv., 2021, 11, 40140-40147.

[0172] [4] Yang, Hee-Man et al, Succinate Functionalization of Hyperbranched Polyglycerol-Coated Magnetic Nanoparticles as a Draw Solute During Forward Osmosis, Journal of Nanoscience and Nanotechnology, Volume 15, Number 10, October 2015, pp . 8279-8284 (6) .

[0173] [5] D. Bartczak et al, Preparation of Peptide- Functionalized Gold Nanoparticles Using One Pot EDC / Sulfo-NHS Coupling, Langmuir 2011, 27, 10119-10123. Analysis :

[0174] DLS

[0175] Aqueous suspensions of conjugates were analyzed with dynamic light scattering (DLS) (Malvern Zetasizer) at final concentrations of 0.02 mg conjugates / mL diluted in water. Suspension homogeneity was assured by ultrasonication for 10 min in an ultrasonication bath during sample preparation and prior to analysis with DLS.

[0176] Zeta -potent! al

[0177] The surface charges of conjugates were evaluated using zeta potential measurements (Malvern Zetasizer) , where the velocity of the particles in an electrical field is recorded using laser Doppler electrophoresis. Aqueous suspensions of conjugates were diluted in 1 mM KC1 to a final concentration of 0.02 mg / mL. Suspension homogeneity was assured by ultrasonication for 10 min in an ultrasonication bath during sample preparation and prior to analysis with DLS.

[0178] AAV9 VC quantification assay

[0179] To quantify the numbers of rAAV9 or rAAV90VCs that were covalently bound to the surface of the particles, a sample of the supernatant of the coupling suspension was taken at the end of the reaction (but before addition of the quencher molecule) and analyzed with an AAV9 quantification ELISA kit (Progen, Cat. No. PRAAV9XP) . To generate the supernatant, the conjugates were magnetically separated. Subtracting the number of capsids in the supernatant from capsids offered to the particles gives the number of capsids bound to the particle surface .

[0180] # (VCs bound to particle surface) =

[0181] # (VCs offered in coupling reaction) - # (VCs left in supernatant after coupling reaction)

[0182] Results : Physico-chemical characterization of conjugates with DLS and Zeta potential measurements

[0183] Two different size fractions were found by DLS: The average hydrodynamic diameter of the first fraction was in the range of 110-143 nm, that of the second fraction remained in the area of 400 to 450 nm.

[0184] Due to the carboxy-groups on the surface of the particles the zeta potential is typically negative, which was confirmed with the result of -37.89 mV. The zeta potential values of the conjugates are in the range between -39 and -33 mV. This is not surprising, as the carboxy groups of the precursor Particle Polymer typically give a negative surface charge, and AAV9 capsids also were reported to exhibit negative zeta potential values. Furthermore, the remaining activated groups on the particles surface where no capsid has bound (due to steric reasons) are quenched with a small molecule which also exhibits carboxy groups.

[0185] Quantification of VCs covalently bound to particles Using the AAV9 VC quantification assay, on average 80% of the VCs were bound per mg particles (Figure 3A) , which corresponds on average to 40% of the offered VCs.

[0186] Example 2: Anti-AAV9 antibody removal

[0187] Example 2.1: Anti-AAV9 antibody removal from PBS using conjugates with rAAV9 VCs Example 2.2: Anti-AAV9 antibody removal from PBS using conjugates with rAAV90VCs

[0188] Materials and Methods:

[0189] Anti-AAV9 antibody binding assay

[0190] A high binding black 384-well microtiter plate (Greiner, 781077) was coated with 1E11 vgc / mL rAAV9 in CMF-DPBS overnight at 4 °C. The plate was washed manually three times with CMF-DPBS, blocked with assay buffer (3 Sigma- Aldrich, Cat. No. A9576) in 1% Casein blocker (Thermo Fisher Scientific, Cat. No. 37528) for 3 hours at RT and washed again 3 times. The samples were added to the plate, incubated for 2 hours at RT, and washed 5 times with PBS. An HRP-conj ugated anti-human Fc detection antibody (Southern Biotech, 9040-05, 0.167 nM) for detection of human anti-AAV9 antibodies, or anti-mouse Fc detection antibody (Jackson ImmunoResearch, 115-035-071, 0.25 nM) for detection of ADK9 was added and incubated for 1 hour at RT in the dark. A QuantaBlu kit (ThermoFisher, 15189) was used as detection method by adding QuantaBlu substrate (1:10) to each well, incubating for 30 minutes at RT in the dark, and lastly adding STOP solution to each well. The plate fluorescent signal was measured in a Tecan Infinite F200 Fluorescence Reader within 30 minutes.

[0191] ADK9 removal assay

[0192] 1 mg / mL conjugates were added to PBS spiked with mouse ADK9 (anti-AAV9 monoclonal antibody, Progen, 610178) at 1 pg / mL. The mixture was incubated for 50 minutes at room temperature under continuous agitation, after which the magnetic conjugates were magnetically separated and the remaining ADK9 was quantified with the "Anti-AAV9 antibody binding assay", an ELISA-based method (see above) . The obtained values were compared to control samples (ADK9-spiked PBS without exposure to conjugates) to determine the relative removal of ADK9 by conjugates.

[0193] Results :

[0194] Determination of ADK9 anti-AAV9 antibody removal capacity of conjugates with rAAV9 or rAAV90

[0195] The goal of this experiment was to determine the anti- AAV9 antibody removal capacity of conjugates (1) in a controlled setting in PBS independent from biological varieties in human plasma and (2) using ADK9, a commercially available anti-AAV9 monoclonal antibody, allowing a quantitative evaluation of the removal capacity of manufactured conjugates using the ADK9 removal assay . The average removal of ADK9 from PBS by conjugates with rAAV9 VCs was in average 45% (SD ± 26%) and for rAAV90VCs was 47% (SD ± 9%) (Figure 3B) .

[0196] Example 2.3: Anti-AAV9 antibody removal from human plasma using conjugates with rAAV9 VCs Example 2.4: Anti-AAV9 antibody removal from human whole blood using conjugates with rAAV9 VCs

[0197] Materials and Methods:

[0198] Conjugate treatment of human plasma or blood The conjugates were applied to remove VC-specific antibodies from blood using a device as described in WO 2021 / 063708 Al. These experiments were performed in a setup like the clinical application (Figure 2) : Conjugates were sonicated and vortexed for at least 30 seconds prior to diluting them in PBS to reach the required concentration used for treating the human plasma. The conjugates with VCs with or without genomic material were then added to the human plasma samples reaching a final concentration of either 2.0 or 0.5 mg / mL. The duration of one treatment cycle was either 3 minutes or 50 minutes at 37 °C under continuous agitation in a HulaMixer Sample Mixer. In some experiments multiple treatment cycles of 3 minutes were performed by magnetically separating the conjugates from the human plasma and adding fresh conjugates for each consecutive treatment cycle. After the last treatment cycle the conjugates were magnetically separated. Both the treated and untreated human plasma was diluted at least 1:3 and the anti-AAV9 antibodies were measured in an ELISA-based method (see above "Anti-AAV9 antibody binding assay") . The values were normalized by correcting for the background the ratio was determined by dividing the measured value by the value of the positive control.

[0199] Results :

[0200] In the clinical setting the conjugate treatment will be performed in whole blood. To ensure that this was feasible, the treatment was performed with EDTA- anticoagulated blood from one donor (cat# 92000, Interregionale Blutspende SRK) containing anti-AAV9 antibodies. The results of the whole blood treatment were comparable to the treatment of human plasma to demonstrate that the treatment in either matrix is comparable. There were no differences in between the conjugate treatment in whole blood versus plasma, proving that the treatment works in both matrices (Figure 4) .

[0201] Example 3: Improvement of gene transduction efficiency after treating human plasma with conjugates

[0202] Example 3.1: Treatment with particle-rAAV9 con ugates Example 3.2: Treatment with particle-rAAV90conj ugates

[0203] Material and Methods :

[0204] Transduction assay

[0205] In the virus neutralization assay the neutralizing antibodies are detected by measuring the transduction efficiency of AAV9 after exposure to anti-AAV antibodies. To determine the virus neutralizing capacities of the antibodies in human plasma before and after conjugate treatment, a virus neutralization assay was performed. HEK293T / 17 HG2 wells were seeded in a tissue culture treated 384-well plate at 8000 cells / well and allowed to attach for 6 hours 37°C + 5% CO2. In the meantime, an 11- point 3-fold serial dilution was prepared with the human plasma, ADK9, and isotype control. A recombinant AAV (GMO RA generic@ACl) containing luciferase was prepared at 4E9 genome copies / mL (multiplicity of infection 5000 on 8000 cells) and the virus was added to the plasma / IgG dilution series. Directly after, the virus / IgG mixture was added to the cells and the plate was incubated for 48 hours at 37 °C + 5% CO2 • The amount of virus in the cells was determined with Steady-Gio. Transduction efficiency after conjugate treatment of human plasma

[0206] To assess if the conjugate treatment would restore transduction efficiency, human plasma was treated with the conjugate and the binding antibodies and transduction efficiency were measured. The treatment was performed with conjugates containing VCs with or without genetic material. This treatment protocol was tested for 3 donors with verified anti-AAV titers. Serum from 3 donors with low titers were used to represent the "gene therapy treatable range", defined as the average value ± SD of these donors in the assay. Treatment with both conjugates led to a reproducible depletion of antibodies that resulted in titers in the same range as the low titer donor samples (Figure 5A) . These depletions correlated with an increased transduction efficiency that was similar for both treatments as well (Figure 5B) . Based on these results it can be concluded that both filled and empty capsids can be used as binding agents to deplete anti-AAV antibodies.

[0207] Example 4 : Synthesis of AAVrh74 VC Conjugates

[0208] The NHS-activated core-shell particles are prepared and washed with PBS and EDTA as described in Example 1. The NHS-activated particle suspension is then immediately added to the previously prepared solution of AAVrh74 VC. Different amounts of AAVrh74 can be added to reach a concentration of AAVrh74 in the range of 0.01-10 mg / mL (e.g. 0.01, 0.05, 0.1, 0.6, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / mL) to alter the coating density of the particles. Optionally, a blocking agent (e.g. human serum albumin, TRIS, glycine) is added to the particle - VC mixture at a molar ratio of VC to blocking agent of 1:100, 1:10, 1:1, 1:0.1, 1:0.01 to avoid or reduce particle aggregate formation. After ultrasonication for 1 min, this reaction mixture is then shaken for at least 3 h at 900 rpm at room temperature. The NHS-activated particles react with free amines on the VCs forming a covalent amide bond. The resulting particles-VC conjugates are sonicated for 30 sec and subsequently magnetically separated. A small sample of the supernatant is removed to determine the concentration of unreacted VC (see section "Quantification of VC bound to particles") . The conjugates are again resuspended using ultrasonication (1 min) and a quenching solution of Tris buffer (20 mM) is added to the suspension. After mixing for 10 minutes at 500 rpm, the conjugates are washed three times with PBS containing 1 mM EDTA, using magnetic separation in between, and then resuspended in PBS at a concentration of 5 mg / mL.

[0209] Example 5: Anti-AAVrh74 Antibody Removal

[0210] Conjugate treatment of human plasma or blood Apply conjugates to remove VC-specific antibodies from blood using a device as described in WO 2021 / 063708 Al. Sonicate and vortex conjugates for at least 30 seconds prior to diluting them in PBS to reach the required concentration used for treatment of human plasma. Add the conjugates with VC (with or without genomic material) to human plasma samples reaching a final concentration between 0.5 - 2.0 mg / mL. Treat the human plasma for 3 or 50 minutes at 37°C under continuous agitation. Multiple treatment cycles are performed, if required, by magnetically separating the conjugates from human plasma and adding fresh conjugates for each consecutive treatment cycle. After the last treatment cycle the conjugates are magnetically separated. Both the treated and untreated human plasma are diluted at least 1:3 and the anti-AAVrh74 antibodies are measured in an ELISA- based method. The values are normalized by correcting for the background and the removal is calculated relative to the positive control (no treatment) .

[0211] Example 6: AAVrh74 Transduction Efficiency Transduction assay

[0212] In the virus neutralization assay the neutralizing antibodies are detected by measuring the transduction efficiency of AAVrh74 after exposure to anti-AAV antibodies. To determine the virus neutralizing capacities of the antibodies in human plasma before and after conjugate treatment, a virus neutralization assay is performed. Cells are seeded in a tissue culture treated plate and attach for 6 hours 37°C + 5% CO2. In the meantime, a serial dilution is prepared with the human plasma, a monoclonal antibody (IgG) against AAVrh74, and isotype control. A recombinant AAVrh74 containing a reporter gene is prepared at the relevant multiplicity of infection and the virus added to the plasma / IgG dilution series. Directly after, the virus / IgG mixture is added to the cells and the plate is incubated for 48 hours at 37 °C + 5% CO2. The amount of virus in the cells is determined by measuring the reporter gene.

[0213] Example 7 : Conjugate size distribution

[0214] Dynamic Light Scattering Three further batches of conjugates were synthesized as described in example 1 and the hydrodynamic diameter was measured using DLS on a BeNano 180 Zeta Pro device. Measurements were performed at 25°C and a detection angle of 173°. In this example, conjugate size varied between about 160 nm and about 220 nm, with a polydispersity index (PDI) of between about 0.16 and 0.27. This experiment confirmed a reproducible conjugate size. Results are shown in table 1 below.

[0215] Table 1. Hydrodynamic diameter Further embodiments of the invention:

[0216] 1. A Conjugate comprising a particle and a multitude of viral structures wherein said particle has a size of 5 nm to 5 micron and is of the core-shell type and comprises:

[0217] • a core, said core consists of an iron - based material having soft magnetic properties;

[0218] • covered by a first shell, said shell contains one or more graphene layers which are optionally functionalized; and / or

[0219] • covered by a second shell, said second shell contains a polymer selected from the group consisting of Polyglycerol, Polyethylene glycol (PEG) , dextran, Hydroxyethyl starch, Polylactic acid (PLA) , Styrene-maleic acid copolymers (SMA) , Polystyrene (PS) , Poly ( ethylene imine) , Chitosan, Polymethylmethacrylate (PMMA) ; with the proviso that, if no second shell is present, said one or more graphene layers are functionalized; with the proviso that the particle shells do not contain magnetic material; and wherein said viral structures:

[0220] • are based on genetically engineered adeno-viruses (AdV) or genetically engineered adeno-associated- viruses (AAV) ; and

[0221] • are selected from viral capsids (VCs) with recombinant genetic material, VCs without recombinant genetic material, individual viral capsid proteins, and / or assemblies of viral capsid proteins . characterized in that said viral structures are covalently bound to said particle shell. The conjugate of embodiment 1, wherein said particle is of the core-shell type and consists of

[0222] Either ('type 1 particles' )

[0223] • a core, said core consists of an iron - based material having soft magnetic properties;

[0224] • covered by said first shell, which contains one or more graphene layers that are functionalized;

[0225] OR ('type 2 particles' )

[0226] • a core, said core consists of an iron - based material having soft magnetic properties;

[0227] • covered by said second shell, said second shell contains or consist of a polymer as defined in claim 1;

[0228] OR ('type 3 particles' )

[0229] • a core, said core consists of an iron - based material having soft magnetic properties;

[0230] • covered by said first shell, which contains one or more graphene layers that are not functionalized; and

[0231] • covered by said second shell, said second shell contains or consist of a polymer as defined in claim 1;

[0232] OR ('type 4 particles' )

[0233] • a core, said core consists of an iron - based material having soft magnetic properties;

[0234] • covered by said first shell, which contains one or more graphene layers that are functionalized; and

[0235] • covered by said second shell, said second shell contains or consist of a polymer as defined in claim 1. The conjugate of embodiment 1 or 2, wherein the core

[0236] • contains an iron-based material which contains at least 50 wt% Fe;

[0237] • has a size of 5 - 150 nm determined by electron microscopy;

[0238] • has soft magnetic properties with a coercive force below 30'000 A / m and a magnetic saturation above 30 emu / g . The conjugate according to any one of embodiments 1 -

[0239] 3, wherein the first shell

[0240] • Comprises 3 - 15 graphene layers; and / or

[0241] • Comprises linkers of formula (I') wherein n is an integer between 1 and 5,

[0242] FG represents independent from each other OR, NHR, NR2, COR, COOR, SR, SOR, SO2R, P(O)OR, and P(O)2OR, R represents independent from each other H, C1-C4 alkyl, C1-C4 alkoxy, C3-C10 cyclo-alkyl, and C4-C10 aryl . The conjugate according to any one of embodiments 1 -

[0243] 4, wherein the polymer of the second shell

[0244] • is a hyperbranched polyglycerol; and

[0245] • is covalently bound to the linker of formula (I') , and

[0246] • comprises linkers selected from the group of thioethers -SR and amides -C(O)NHR where R represents of C1-C4 alkyl, C1-C4 alkyloxy, or an amino acid; and

[0247] • preferably where the polymer accounts for 10 wt% - 60 wt% of the particle including second shell. The conjugate according to any one of embodiments 1 -

[0248] 5, wherein said viral structures are based on recombinant adeno-viruses (rAdV) or recombinant adeno-associated-viruses (rAAV) ; preferably rAAV. The conjugate according to any one of embodiments 1 -

[0249] 6, wherein said viral structures are VCs . The conjugate according to any one of embodiments 1 -

[0250] 7, comprising • a particle with a hydrodynamic diameter of 5 - 600 nm ( as determined by Dynamic Light Scattering ( DLS ) ) ,

[0251] • 2 - 2000 viral structures covalently bound to the surface of said particles ( as quanti fied by the concentration determination of unbound viral structures that remain in the liquid phase after the coupling reaction) ; and / or

[0252] • has a zeta potential < 0 mV . A liquid formulation comprising conj ugates according to any of embodiments 1 - 8 and a dispersion medium, wherein :

[0253] • the dispersion medium being selected from pharmaceutically acceptable aqueous media, preferably buf fered aqueous media ; and / or

[0254] • the concentration of said conj ugates being in the range of 20 - 200 mg / mL ; and / or

[0255] • the pH of the formulation being in the range 5- 9 ;

[0256] • the osmolality of the formulation being in the range of 200 - 400 mOsm / kg . The liquid formulation according to embodiment 9 comprising either

[0257] • a multitude of one type of conj ugates ; or

[0258] • a multitude of two or more types of conj ugates . The conj ugate of any one of embodiments 1 - 8 for use in the removal of antibodies from blood, wherein the antibodies being selected from the group consisting of anti-AAV antibodies and anti-AdV antibodies . A method for removing antibodies from blood, the antibodies being selected from the group consisting of anti-AAV antibodies and anti-AdV antibodies , the method comprising : a. contacting the blood with a conjugate according to any of embodiments 1 - 8, or a formulation according to embodiments 9 - 10, b. mixing the thus obtained composition to thereby effect binding of said antibodies to the conjugate to obtain an antibody-conjugate-complex, c. separating the antibody-conjugate-complex from the blood by means of a magnetic material. The method according to embodiment 12, or the conjugate for use according to embodiment 11, wherein

[0259] • the antibody is an anti-AAV antibody; and / or

[0260] • said blood is whole blood or blood plasma or blood serum; and / or

[0261] • a titer of said antibody in the blood, measured within 24 hrs after removal, is reduced to less than 50% of the initial titer; and / or

[0262] • wherein said removal of antibodies involves (i) continuous removal of blood from a patient and (ii) continuous returning of blood which is depleted from said antibodies, to said patient, over a period of 0.5 - 5 hrs; and / or

[0263] • wherein said removal is performed in one to five sessions . The conjugate of any one of embodiments 1 - 8 for use in AdV- or AAV based Gene therapy. A method of administering a viral vector to a patient the vector being selected from genetically engineered AAV vectors and genetically engineered AdV vectors, preferably rAAV and rAdV vectors; the method comprising:

[0264] • in a first step removing the subject's circulating antibodies by the method according to embodiment 12 or 13;

[0265] • in a second step administering the viral vector to the subject; • optionally repeating said first and said second step for 1 - 10 times. The method of embodiment 15, wherein: • the second step is performed within 1 hour - 7 days after completing the first step; and / or

[0266] • wherein the viral vector is a vehicle for Gene therapy; and / or

[0267] • wherein the viral vector is an rAAV vector.

Claims

Claims1. A Conjugate comprising a particle and a multitude of viral structures wherein said particle has a size of 5 nm to 5 pm and is of the core-shell type and comprises:• a core consisting of an iron - based material having soft magnetic properties;• covered by a first shell comprising one or more graphene layers, optionally wherein the graphene layers are functionalized; and / or• covered by a second shell comprising a polymer selected from the group consisting of Polyglycerol, dextran, Hydroxyethyl starch, Polylactic acid (PLA) , Styrene-maleic acid copolymers (SMA) , Polystyrene (PS) , Poly ( ethylene imine) , Chitosan, Polymethylmethacrylate (PMMA) ; with the proviso that, if no second shell is present, said one or more graphene layers are functionalized; with the proviso that the particle shells do not contain magnetic material; and wherein said multitude of viral structures:• are covalently bound to said particle;• are based on genetically engineered adeno-viruses (AdV) or genetically engineered adeno-associated- viruses (AAV) ; and• are selected from viral capsids (VCs) with recombinant genetic material, VCs without recombinant genetic material, individual viral capsid proteins, and / or assemblies of viral capsid proteins, preferably VCs with recombinant genetic material or VCs without recombinant genetic material, more preferably VCs without recombinant material .

2. The conjugate according to claim 1, wherein• 2 - 2000 of the viral structures are covalently bound to the particle shell, preferably 2 - 100, more preferably 2-50; and / or• a ratio of particle : viral structures is between 1 : 2 and 1 : 2000, preferably between 1:2 and 1:100, more preferably between 1:2 and 1:50.

3. The conjugate according to claim 1 or 2, wherein• the particle size is between 20 nm - 5 pm, preferably between 20 nm - 150 nm, more preferably between 40 nm - 100 nm, as determined by electron microscopy, and / or• the particle has a hydrodynamic diameter of 5 nm- 600 nm, preferably 100 nm - 300 nm, as determined by Dynamic Light Scattering (DLS) , and / or• the conjugate has a hydrodynamic diameter of 5 nm- 600 nm, preferably 100 nm - 600 nm, more preferably 100 nm - 300 nm as determined by Dynamic Light Scattering (DLS) , and / or• the conjugate has a hydrodynamic diameter of 20 nm - 300 nm, preferably 35 nm - 200 nm, more preferably 50 nm - 100 nm as determined by analytical centrifugation.

4. The conjugate according to any one of claims 1 to 3, wherein said core-shell type particle consists of either ('type 1 particles' )• the core consisting of an iron - based material having soft magnetic properties;• covered by said first shell comprising one or more graphene layers, wherein the one or more graphene layers are functionalized;OR ('type 2 particles' )• the core consisting of an iron - based material having soft magnetic properties;• covered by said second shell, wherein said second shell comprises or consists of a polymer as defined in claim 1;OR ('type 3 particles' )• the core consisting of an iron - based material having soft magnetic properties;• covered by said first shell comprising one or more graphene layers, wherein the one or more graphene layers are not functionalized; and• covered by said second shell, wherein said second shell contains or consists of a polymer as defined in claim 1;OR ('type 4 particles' )• the core consisting of an iron - based material having soft magnetic properties;• covered by said first shell comprising one or more graphene layers, wherein the one or more graphene layers are functionalized; and• covered by said second shell, wherein said second shell contains or consists of a polymer as defined in claim 1.

5. The conjugate according to any one of claims 1 to 4, wherein the core complies with one or more of the following characteristics:• containing an iron-based material comprising at least 50 wt% iron; and / or• comprising 10 wt% - 100 wt% iron carbide, preferably 20 wt% - 100 wt%, more preferably 50 wt%- 100 wt%; and / or• comprising 5 wt% - 90 wt% elemental iron (Fe(0) ) , preferably 10 wt% - 40 wt%, more preferably 10 wt%- 30 wt%; and / or• comprising a total content of iron carbide and elemental iron of between 90 wt% - 100wt% of the core mass, preferably 95 wt% - 100 wt%, more preferably 98 wt% - 100 wt%; and / or• comprising less than 5 wt% of iron oxide, preferably comprising less than 1 wt% of iron oxide; and / or• has a size of 5 nm - 150 nm, preferably 10 nm - 50 nm, more preferably 20 nm - 50 nm determined by electron microscopy; and / or• has soft magnetic properties with a coercive force below 30'000 A / m and a magnetic saturation above 30 emu / g .

6. The conjugate according to any one of claims 1 to 5, wherein the first shell• Comprises 3 - 15 graphene layers; and / or• Comprises linkers of formula (I')(I' ) wherein the squiggly line represents a graphene layer, n is an integer between 1 and 5,FG represents independent from each other OR, NHR,NR2, COR, COOR, SR, SOR, SO2R, P(O)OR, and P(O)2OR, R represents independent from each other H, C1-C4 alkyl, C1-C4 alkoxy, C3-C10 cyclo-alkyl, and C4-C10 aryl .

7. The conjugate according to any one of claims 1 to 6, wherein the polymer of the second shell is polyglycerol, preferably hyperbranched polyglycerol.

8. The conjugate according to any one of claims 1 to 7, wherein the particle comprises the second shell, and wherein the polymer• is a hyperbranched polyglycerol; and• is covalently bound to the linker of formula (I') , and• comprises a second group of linkers, preferably comprising functional groups selected from the group consisting of thioethers and amides; more preferably said linkers are selected from the group consisting of thioethers -SR and amides -C(O)NHR where R represents C1-C4 alkyl, C1-C4 alkyloxy, or an amino acid; and• preferably wherein the polymer accounts for 10 wt% - 60 wt% of the particle including the second shell .

9. The conjugate according to any one of claims 1 - 8, wherein said viral structures are based on recombinant adeno-viruses (rAdV) or recombinant adeno-associated-viruses (rAAV) ; preferably rAAV.

10. The conjugate according to any one of claims 1 - 9, wherein said viral structures are VCs .

11. The conjugate according to any one of claims 1 - 10 having a zeta potential < 0 mV.

12. The conjugate according to any one of claims 1 to 11, wherein the viral structures are viral capsids based on AAV selected from the group consisting of AAVrh74, AAV9, AAV8, AAV2 , AAV5, and myotropic AAVs, such as AAVMYO and myoAAV, preferably selected from AAVrh74 and AAV9.

13. A liquid formulation comprising conjugates according to any one of claims 1 to 12 and a dispersion medium, wherein :• the dispersion medium being selected from pharmaceutically acceptable aqueous media, preferably buffered aqueous media; and / or• the concentration of said conjugates being in the range of 20 - 200 mg / mL; and / or• the pH of the formulation being in the range 5-9;• the osmolality of the formulation being in the range of 200 - 400 mOsm / kg.

14. The liquid formulation according to claim 13 comprising either• a multitude of one group of conjugates; or• a multitude of two or more groups of conjugates, wherein the groups of conjugates comprise the same particle and differ in the viral structure.

15. The liquid formulation according to claim 13 or 14, wherein a ratio of particle : viral structures is between 1:2 and 1:2000, preferably between 1:2 and 1:1000, more preferably between 1:2 and 1:50.

16. The liquid formulation according to any one of claims 13 to 15, wherein the size distribution of the conjugates is between 60 nm - 300 nm, preferably between 100 nm - 300 nm as measured by dynamic light scattering, preferably wherein a polydispersity index is between 0.01 - 0.3.

17. The conjugate according to any one of claims 1 to 12 for use in the removal of antibodies from blood, wherein the antibodies are selected from the group consisting of anti-AAV antibodies and anti-AdV antibodies .

18. A method for removing antibodies from blood, the antibodies being selected from the group consisting of anti-AAV antibodies and anti-AdV antibodies , the method comprising: a. contacting the blood with a conjugate according to any one of claims 1 - 12, or a formulation according to any one of claims 13 - 16,b. mixing the thus obtained composition to thereby effect binding of said antibodies to the conjugate to obtain an antibody-con ugate-complex, c. separating the antibody-conjugate-complex from the blood by means of a magnetic material.

19. The method according to claim 18, or the conjugate for use according to claim 17, wherein• the antibody is an anti-AAV antibody; and / or• said blood is whole blood or blood plasma or blood serum; and / or• a titer of said antibody in the blood, measured within 24 hrs after removal, is reduced to less than 50% of the initial titer; and / or• wherein said removal of antibodies involves (i) continuous removal of blood from a patient and (ii) continuous returning of blood which is depleted from said antibodies, to said patient, over a period of 0.5 - 5 hrs; and / or• wherein said removal is performed in one to five sessions .

20. The conjugate according to any one of claims 1 to 12 for use in AdV- or AAV based Gene therapy.

21. The conjugate for use according to claim 20, wherein the gene therapy is in vivo CRISPR-Cas gene editing.

22. A method of administering a viral vector to a patient the vector being selected from genetically engineered AAV vectors and genetically engineered AdV vectors, preferably rAAV and rAdV vectors; the method comprising:• in a first step removing the subject's circulating antibodies by the method according to claim 18 or 19;• in a second step administering the viral vector to the subject;• optionally repeating said first and said second step for 1 - 10 times.

23. The method of claim 22, wherein: • the second step is performed within 1 hour - 7 days after completing the first step; and / or• wherein the viral vector is a vehicle for Gene therapy; and / or• wherein the viral vector is an rAAV vector.

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