Broad spectrum virus capture nanoshell

Abstract

The present invention relates to a DNA-based nanostructure for encapsulating a virus or a viral particle, to a composition comprising one or more viruses or viral particles encapsulated by such a DNA-based nanostructure according to the invention, and to a method for encapsulating one or more viruses or viral particles by using such DNA-based nanostructures.

Description

Technical Field

[0001] The present invention relates to a DNA-based nanostructure for encapsulating a wide range of viruses or virus particles, to a composition comprising one or more viruses or virus particles encapsulated by such a DNA-based nanostructure according to the present invention, and to a method for encapsulating one or more viruses or virus particles by using such a DNA-based nanostructure. Background Art

[0002] Currently, more than 200 human diseases transmitted by viral vectors are known, of which only nine can be treated with existing antiviral drugs (Heida et al., Drug Discov. Today 26 (2021) 122–137). In the search for effective antiviral therapies, neutralizing antibodies are increasingly being considered for the treatment of acute viral infections (see, for example, Wang et al., Science. August 13, 2021; 373 (6556): eabh1766. doi: 10.1126 / science.abh1766. July 1, 2021 electronic version. PMID: 34210892; Taylor, PC et al., Nat. Rev. Immunol. 21 (2021) 382–393). Antiviral antibodies generally obtain virus neutralization function by blocking the interaction of viruses with specific receptors on the surface of host cells, and the blocked interaction is required for receptor-mediated cell invasion. However, antibodies are prone to losing their function due to mutational drift, take time to develop, and are only effective against one virus or viral serotype at a time. In addition, antibodies or other protein-virus binders may cause adverse immunogenic effects in organisms and create significant additional manufacturing hurdles and costs.

[0003] Recently, a new concept for neutralizing viruses by encapsulation in macromolecular shells made using DNA origami has been proposed (WO 2021 / 165528). The shell mechanically prevents the interaction between the captured virus and the host cell. For proof-of-concept experiments, the interior of the shell was coated with antibodies to sequester the virus particles in the shell. Heparin was mentioned as a potential alternative binding moiety, but no data was shown. A key advantage of the shell is that the virus binding moiety used inside it does not itself need to have a neutralizing function, as this task is performed by the shell material. Nevertheless, as mentioned above, the use of antibodies in virus capture shells poses some challenges that may limit the usefulness of the virus capture concept. WO 2021 / 165528 uses up to 90 antibodies per virus capture shell, which are connected for hybridization via interaction with a single-stranded oligonucleotide handle with a 16-mer or 26-mer overhang. WO 2021 / 165528 demonstrated that virus particles can be successfully encapsulated in shells equipped with antibodies, but the encapsulation rate achieved was not quantified.

[0004] The concepts proposed in WO 2021 / 165528 have also been described in a scientific publication (Sigl et al., Nat. Mater. 20 (2021) 1281-1289). Sigl et al. demonstrated successful encapsulation of viral particles using antibody-equipped viral particles. Heparin was not mentioned as an alternative binding moiety, and no quantification of encapsulation efficiency was provided.

[0005] Knappe et al. (ACS Nano 2021; 14316-14322) describe DNA origami particles functionalized via click chemistry, allowing different types of functional moieties (including antibodies and carbohydrates) to be coupled to the DNA origami particles. Knappe et al. do not specifically mention heparin, and the functionalization is performed on the outside of the DNA origami particles because the DNA origami particles are closed shells. Therefore, no information can be obtained from Knappe et al. regarding the options for encapsulating viral particles inside the DNA origami shells and any details on the optimal design of the attachment sites.

[0006] Another attractive approach is to package viral vectors to deliver the genetic information or other cargo of the virus or other vector to the target cell or target tissue, as discussed, for example, in Antigen-Triggered Logic-Gating of DNA Nanodevices, Engelen et al., J. Am. Chem. Soc. 2021, 143, 51, 21630–21636, Dec. 20, 2021. In addition, due to the challenges of using antibodies listed above, the use of antibodies as part of attaching viruses or virus-like particles or other assemblies within a DNA-based shell may limit the scope of such applications.

[0007] Therefore, despite the different strategies that have been developed or suggested to date for the treatment of viral infections, there is still a need for concepts that develop universal antiviral drug platforms against multiple viral pathogens. In particular, a concept that does not rely on detailed prior knowledge about the genetics and properties of the target virus is highly desirable. Furthermore, there is an unmet need for the development of systems that allow for the efficient encapsulation of viral particles. Summary of the invention

[0008] It is an object of the present invention to provide constructs capable of encapsulating one or more viruses or viral particles.The prior art has not taught or suggested a solution to this problem, namely the use of macromolecular building blocks, such as DNA-based nanostructures.

[0009] Therefore, in one aspect, the present invention relates to a DNA-based nanostructure, wherein the DNA-based nanostructure is a shell comprising a cavity surrounded by the DNA-based nanostructure, wherein the DNA-based nanostructure is formed by self-assembling DNA-based structural units, wherein each of the self-assembling DNA-based structural units is formed by a single-stranded DNA template chain and a set of oligonucleotides complementary to the single-stranded DNA template, wherein each of the oligonucleotides is complementary to a continuous DNA sequence stretch or at least two non-continuous DNA sequence stretches on the single-stranded DNA template, wherein each of the self-assembling DNA-based structural units is complementary to a continuous DNA sequence stretch or at least two non-continuous DNA sequence stretches on the single-stranded DNA template. One is a triangular prism and / or a rectangular prism, in particular a triangular prism, and wherein a subset of one or more of the oligonucleotides in one or more of the self-assembling DNA-based structural units is each connected to a construct comprising at least one sulfonated or sulfated polysaccharide group pointing to the interior of the cavity, in particular a construct comprising one or two sulfonated or sulfated polysaccharide groups, wherein each of the constructs comprises (i) a handle comprising at least one binding site for the sulfonated or sulfated polysaccharide group, and (ii) the sulfonated or sulfated polysaccharide group bound to the handle; wherein the handle has a length at least corresponding to the length of a single-stranded oligonucleotide comprising 30 nucleotides.

[0010] In a second aspect, the present invention relates to a composition comprising a DNA-based nanostructure according to the present invention encapsulating one or more viruses or viral particles.

[0011] In a third aspect, the present invention relates to a method for encapsulating one or more viruses or viral particles, comprising the steps of providing a DNA-based nanostructure according to the present invention, and contacting the DNA-based nanostructure with a medium containing or suspected of containing the virus or viral particles.

[0012] The present disclosure encompasses all combinations of any one or more of the foregoing aspects and / or embodiments, as well as combinations with any one or more of the embodiments set forth in the Detailed Description and Examples.

[0013] Other features, objects, and advantages of the compositions and methods herein will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1DNA origami shells and functionalization with HS derivatives are shown. A: Heparan sulfate proteoglycans (HSPGs) interact with viral pathogens and mediate their cellular uptake (left). Schematic of DNA origami shells with HS modifications in their interior that are able to bind and sequester viral particles (right). B: SPAAC reaction between azide-modified HS oligomers and DBCO-modified DNA oligonucleotides. The DNA sequences are complementary to the handles of the DNA origami shells in (e,f). C: PAGE characterization of HS-modified DNA oligonucleotides. Products containing HS with sulfate and sulfonate groups (3a and 3c) migrate through the gel at a faster rate than similar negative controls (3b and 3d) due to their enhanced anionic properties. D: Cylindrical models of O and T1 shells are composed of 4 and 10 triangular subunits, respectively, containing single-stranded protruding oligonucleotides (called handles, shown in red) decorating their interiors. Each triangular subunit contains 9 handle positions. E: A new T3 shell design consisting of 30 triangular subunits with a 150 nm inner cavity. Each triangular subunit also contains 9 handle positions. F: Negative stain TEM micrograph of the T3 shell. Scale bar 100 nm. G: Schematic diagram of three different handle designs. H1 contains one HS modification per handle, placed as close as possible to the origami surface. H2 also contains one HS modification per handle, but has a 20-base polyT stretch, allowing the handle to reach further than H1. H3 simulates a branched polymer with two HS modifications per handle unit, thus doubling the local HS density.

[0015] Figure 2 Viruses and VLPs captured in HS-modified O, T1, and T3 capsids are shown. Negative stain TEM illustrations of: a: AAV2, poliovirus type 3, mature dengue virus type 1, and GII.4 norovirus successfully captured in O capsids; b: HPV 16, SARS-CoV-2, chikungunya virus, and rubella virus entrapped in T1 capsids; c: Adenovirus type 5 captured with T3 capsids. Scale bar 100 nm.

[0016] Figure 3 Multiple viruses and VLPs captured in HS-modified O, T1, and T3 capsids are shown. Negative stain TEM images of: a: up to four AAV2s in one O capsid, b: up to three HPV 16s in one T1 capsid, c: coordinated capture of one HPV 16 by two O capsids to completely enclose the virus particle; d: up to six AAV2s per T1 capsid; e: up to three Chikungunya virus VLPs per T3 capsid; f: synergistic effect of multiple O capsids capturing a large number of AAV2 particles. Scale bar 100 nm.

[0017] Figure 4 Cryo-EM analysis of virus-like particles captured in DNA origami shells is shown. a: Cryo-EM micrograph of O shell bound to HPV 16 VLP; b: 2D class-averaged images of one or two O shells bound to one HPV 16 particle, showing different orientations of the complex. The white arrow indicates the difference in the gap between the two O shells, confirming the capture of VLP particles of different sizes; c: 3D reconstruction of HPV 16 bound to one and two O shells; d: Cryo-EM micrograph of T1 shell bound to Chikungunya virus VLP; e: 2D class-averaged images of T1 shell bound to Chikungunya virus particles, showing different orientations of the complex; f: Two different views of the 3D reconstruction of T1 shell entrapped Chikungunya virus particles.

[0018] Figure 5 SPAAC reactions of 8-mer HS derivatives (1a and 1b) are shown. a: Click chemistry reaction between azide-modified HS polymers and DBCO-modified DNA oligonucleotides. The DNA sequence is complementary to the handle of the DNA origami shell. b: PAGE characterization of all HS-modified DNA oligonucleotides.

[0019] Figure 6 The T3 shell design is shown. a: Top and front views of the T3 cylindrical model. b: Cylindrical model of triangles t1-t6 involved in the T3 shell assembly. Arrows indicate complementary side interactions.

[0020] Figure 7 A TEM field of view of a T3 shell is shown. The T3 DNA origami shells exhibit an inner diameter of approximately 150 nm. Due to their flexibility, they appear deformed on the grid. The scale bar is 400 nm.

[0021] Figure 8 TEM quantification results of O shells for AAV2 capture using different handle designs are shown. a: Schematic diagram of three different handle designs H1, H2 and H3. HS are represented as red hexagons. H1 contains one HS modification per handle and is placed close to the origami surface. H2 also contains one HS modification per handle, but has a 20-base polyT extension, allowing the handle to reach further than H1. H3 mimics a branched polymer containing two HS modifications per handle unit, thus doubling the local HS density. b: Blind TEM quantification of full and empty O shells for each handle design when functionalized with 3c HS derivatives and AAV2 in excess. H1 shows about 20% full shells, H2 about 84%, and H3 about 96%. c: Schematic diagram of the O half shells and the ssDNA handles in their lumen.

[0022] Fig. 9 TEM of a negative control for AAV2 capture in O capsid is shown. Two fields of view of the same sample show that no binding was observed when the 3d negative control HS modification was hybridized to the H3 handle design. Scale bar is 100 nm.

[0023] Fig.10 TEM of AAV2 trapped using excess O capsids is shown. Two fields of view of the same sample show that all AAV2 particles are encapsulated when origami capsids are used in excess. Scale bar is 100 nm.

[0024] Fig.11 TEM of free virus and VLPs are shown. TEM data show that AAV2, poliovirus, HPV 16, chikungunya virus and adenovirus type 5 are the purest samples in our library. Dengue virus, norovirus, SARS-CoV-2 and rubella virus clearly contain a large amount of protein fragments and show different particle size ranges. The scale bar is 100nm.

[0025] Fig.12 TEM tomograms of adenovirus type 5 in T3 capsids are shown. Slices of tomograms calculated from EM tilt series demonstrate complete encapsidation of adenovirus in selected capsid particles. Scale bar 100 nm.

[0026] Fig.13 TEM tomograms of Chikungunya virus VLPs in T3 capsids are shown. Slices of the tomogram calculated from the EM tilt series demonstrate complete encapsidation of three Chikungunya virus VLPs in the selected capsid particle. The last image slice shows that the connectivity of the triangles is disrupted. It is unclear whether this discontinuity is a result of the capsid rearrangement to encapsidate multiple VLPs or a result of deformation on the grid during sample preparation. Scale bar 100 nm.

[0027] Fig.14 TEM quantification of T1 capsids captured by 3a and 3b HS derivatives of Chikungunya virus VLPs is shown. a: Negative stain TEM micrograph of T1 capsids functionalized with 3b negative control HS derivative. Due to size and shape complementarity, weak electrostatic interactions between DNA and Chikungunya virus VLPs are sufficient to keep the virus particles encapsulated when the negative control handle is used. Scale bar is 100 nm. b: TEM quantification of full versus empty T1 capsids functionalized with 3a HS derivative on H1 handle design. Approximately 90% of the capsids are full. c: TEM quantification of full versus empty T1 capsids functionalized with 3b negative control HS derivative on H1 handle design. Approximately 54% of the capsids are full.

[0028] Fig.15TEM of immature and mature dengue virus type 1 VLPs captured with O capsids are shown. a: Immature conformation of dengue virus VLPs showing no binding to HS-modified origami capsids. b: Mature dengue virus VLPs recognized and encapsidated by O capsids. VLPs were used in excess. Scale bar 100 nm.

[0029] Fig.16 Cryo-EM imaging of O shells capturing HPV 16 particles (EMD-13884) is shown. a: Exemplary micrograph of O shells capturing HPV 16 vitrified on a lacey carbon grid with an ultrathin carbon support. b: 2D class averages of empty shells (left), HPV 16 captured by one O shell (middle), and two O shells capturing HPV 16 (right). c: 3D classes of selected selected particles showing similar particles as in b. d: 3D reconstruction of an HPV 16 particle captured in one O shell. e: Multi-body refinement of an HPV 16 particle encapsulated by two O shells. f+g: Multi-component analysis of two O shells capturing HPV 16.

[0030] Fig.17 Cryo-EM imaging of T1 capsids of captured Chikungunya virus VLPs (EMD-13883) is shown. a: Exemplary micrograph of T1 capsids of captured Chikungunya virus VLPs vitrified on lacey carbon grids with ultrathin carbon support. b: 2D class averages of extracted particles. c: FSC estimates of the reconstruction shown in e (C5). d: 3D classification of extracted particles. e: 3D reconstruction of T1 capsids of captured Chikungunya virus of selected particles from multiple rounds of 3D classification, without symmetry (C1) and with symmetry (C5).

[0031] Fig.18 2D class averages of free HPV 16 VLPs extracted from cryo-EM images are shown. The diameters of HPV 16 VLPs range from 35 nm to 50 nm.

[0032] Fig.19 The stability of capsid-captured virus is shown. AAV2 captured in O capsids was quantified by TEM after a dilution series. The fraction of occupied capsids remained the same before and after a 100-fold dilution and incubation at RT for 14 days relative to the undiluted sample. The total capsid concentration was 0.072 nM.

[0033] Fig. 20 TEM of Rubella virus protein fragments trapped in T1 capsid is shown. Protein fragments in Rubella virus VLP samples were successfully encapsulated.

[0034] Fig.21Results of negative staining TEM imaging of virus mixtures captured with heparan sulfate modified T1 hemicapsids are shown. (A, B) TEM fields of T1 hemicapsids capturing AAV2, Chikungunya, and HPV16 virus particles at different ratios, with homogeneous and heterogeneous complexes. Selected captured virus particles are (C) one Chikungunya, (D) one HPV16, (E) one AAV2, (F top) several AAV2, (F bottom) HPV16-Chik, (G) AAV2-Chik, (H) AAV2-HPV16. All scale bars: 100 nm.

[0035] Fig. 22 Shown is the synergistic effect of multiple O capsids capturing a large number of AAV2 particles. Scale bar: 50 nm. DETAILED DESCRIPTION

[0036] The present disclosure provides constructs capable of encapsulating viruses or viral particles.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0038] Unless otherwise indicated, the terms "comprising" and "including" are used herein in their open and non-limiting sense. For such latter embodiments, the term "comprising" therefore includes the narrower term "consisting of."

[0039] Unless otherwise indicated herein or clearly contradicted by the context, in the context of describing the present invention (especially in the context of the appended claims), the terms "a / an" and "the" and similar referents should be understood to cover both the singular and the plural. For example, the term "cell" includes a plurality of cells, including mixtures thereof. When the plural form is used for compounds, salts, etc., this is also meant to be a single compound, salt, etc.

[0040] Therefore, in one aspect, the present invention relates to a DNA-based nanostructure, wherein the DNA-based nanostructure is a shell comprising a cavity surrounded by the DNA-based nanostructure, wherein the DNA-based nanostructure is formed by self-assembling DNA-based structural units, wherein each of the self-assembling DNA-based structural units is formed by a single-stranded DNA template chain and a set of oligonucleotides complementary to the single-stranded DNA template, wherein each of the oligonucleotides is complementary to one continuous DNA sequence segment or at least two non-continuous DNA sequence segments on the single-stranded DNA template, wherein the self-assembling Each of the DNA-based building blocks is a triangular prism and / or a rectangular prism, in particular a triangular prism, and wherein a subset of one or more of the oligonucleotides in one or more of the self-assembling DNA-based building blocks is each linked to a construct comprising at least one sulfonated or sulfated polysaccharide group directed to the interior of the cavity, in particular a construct comprising one or two sulfonated or sulfated polysaccharide groups, wherein each of the constructs comprises (i) a handle comprising at least one binding site for the sulfonated or sulfated polysaccharide group, and (ii) the sulfonated or sulfated polysaccharide group bound to the handle.

[0041] In an alternative embodiment, a subset of one or more of said oligonucleotides in one or more of said self-assembling DNA-based building blocks is linked to a construct comprising at least one sialic acid group, in particular a construct comprising one or two sialic acid groups, directed towards the interior of said cavity.

[0042] In certain embodiments, the handle has a length that at least corresponds to the length of a single-stranded oligonucleotide comprising 30 nucleotides.

[0043] By using such extended forms of linking moieties, surprisingly, a significant increase in the number of DNA-based nanostructures that could be demonstrated to actually encapsulate viral particles could be demonstrated.

[0044] In the context of the present disclosure, the term "DNA-based nanostructure" refers to a nanostructure formed by a group of DNA-based macromolecules. DNA-based nanostructures of the type used according to the present invention are described in detail in WO 2021 / 165528 and Sigl et al. (cited above).

[0045] In the context of the present disclosure, the term "DNA" refers to a deoxyribonucleic acid composed of single-stranded monomeric units (called nucleotides), wherein each nucleotide consists of a nitrogenous nucleobase, a 2-deoxyribose moiety and a phosphate group, wherein the individual nucleotides are linked in the single strand by the phosphate group, which connects the OH group at the 5' position of the 2-deoxyribose moiety to the OH group at the 3' position of the adjacent 2-deoxyribose moiety. In a specific embodiment, the nitrogenous nucleobase is independently selected from cytosine [C], guanine [G], adenine [A] and thymine [T]. In a particular embodiment, one or more of the nucleobases is a non-canonical base, in particular a non-canonical base selected from the following list: a modified adenosine, in particular N6-carbamoyl-methyladenine or N6-methyladenine; a modified guanine, in particular 7-deazaguanine or 7-methylguanine; a modified cytosine, N4-methylcytosine, 5-carboxycytosine, 5-formylcytosine, 5-glycosylhydroxymethylcytosine, 5-hydroxycytosine or 5-methylcytosine; a modified thymidine, in particular α-glutamylthymidine or α-putrescylthymidine; uracil or a modification thereof, in particular uracil, base J, 5-dihydroxypenturacil; or 5-hydroxymethyldeoxyuracil; deoxyuracil and 2,6-diaminopurine. A fragment of a single-stranded DNA can interact with a complementary fragment of DNA through the interaction of complementary nucleobases, wherein cytosine and guanine and adenine and thymine complement each other by forming two (A / T) and three (G / C) hydrogen bonds between the nucleobases, respectively. Two single-stranded DNAs can be fully complementary to each other, as in the case of genomic DNA, or partially complementary to each other, including the case where one single-stranded DNA is partially complementary to two or more other single-stranded DNA strands. The interaction of two complementary single-stranded DNA sequences results in the formation of a double-stranded DNA double helix.

[0046] As is well known, DNA evolved in nature as a carrier of genetic information encoding proteins. DNA also includes non-coding regions, which include regions with regulatory functions. Therefore, any DNA-based application is usually strictly dependent on a specific DNA sequence and can almost always only be enabled by naming a specific DNA sequence. In contrast, in the context of the present invention, such coding and / or regulatory functions do not play any role and may or may not exist, because the basic DNA sequence is designed and selected only in a manner to form the desired arrangement of double-helical subunits. Therefore, in one embodiment, any form of long single-stranded DNA sequence can be selected as a template, whether it is naturally occurring DNA (such as phage DNA) or synthetically produced DNA, and a set of short single-stranded DNA sequences can be designed, wherein each sequence is complementary to one or more different parts of the template and thus forms one or more double-helical parts. In general, all of these double-helical parts are generated by the interaction of a full set of short single-stranded DNA sequences with the template, and then form the desired three-dimensional arrangement. Starting from a given single-stranded template sequence, a set of complementary sequence designs can be set using known techniques, for example, such as the method described for synthesizing a megadalton-class discrete object with a well-defined 3D shape (18, 24-35). In particular, iterative design combined with caDNAno (37) and elastic network-guided molecular dynamics simulations (38) can be used.

[0047] In addition to the interaction of complementary nucleobases of different segments of single-stranded DNA via hydrogen bonds, other interactions may exist between different DNA strands, including stacking interactions between the flat ends of two double-stranded DNA helices (36), thereby enabling the design and formation of complex DNA-based nanostructures via shape complementarity of double-helical subunits. Thus, the two three-dimensional arrangements formed according to the preceding paragraphs may interact with each other by stacking interactions between double-helical subunits present on the two three-dimensional arrangements, including specific interactions between two three-dimensional arrangements having complementary protrusions and recesses (or knobs and holes), for example, as described in WO 2021 / 165528. Figure 7-13 As shown in D.

[0048] In an alternative aspect of the invention, the present invention relates to macromolecule-based nanostructures, which are RNA-based nanostructures.

[0049] In the context of the present disclosure, the term "RNA" refers to a ribonucleic acid composed of a single-stranded monomeric unit (called a nucleotide), wherein each nucleotide is composed of a nitrogenous nucleobase, a ribose moiety, and a phosphate group, wherein each nucleotide is connected in a single strand by a phosphate group, and the phosphate group connects the OH group at the 5' position of the ribose moiety to the OH group at the 3' position of the adjacent ribose moiety. In a particular embodiment, the nitrogenous nucleobase is independently selected from cytosine [C], guanine [G], adenine [A], and uracil [U]. In a particular embodiment, one or more of the nucleobases are non-standard bases, particularly non-standard bases selected from the following list: pseudouridine, ribothymidine, and inosine. Unlike DNA, RNA is usually in a single-stranded form, but may form a double-stranded form by the interaction of complementary nucleobases, wherein cytosine and guanine and adenine and uracil complement each other by forming two (A / U) and three (G / C) hydrogen bonds between the nucleobases, respectively.

[0050] In the context of the present invention, the term "cavity" refers to the space surrounded by the DNA-based nanostructure. In a specific embodiment, the cavity is similar to a sphere, wherein the spherical part has been cut off, wherein the cutting plane is formed by the self-assembled DNA-based structural units at the boundary of the DNA-based nanostructure. In a specific embodiment, the cutting plane is a great circle, so that the DNA-based nanostructure is a half shell. In other cases, when the three-dimensional geometry of the DNA-based nanostructure is derived from a spherocylinder or a polyhedron, in particular a tetrahedron, an octahedron or an icosahedron, the cavity is understood to be a space formed by cutting the corresponding spherocylinder or polyhedron using a plane, wherein the cutting plane is formed by the self-assembled DNA-based structural units at the boundary of the DNA-based nanostructure.

[0051] In certain other embodiments, the DNA-based nanostructure resembles a spherical portion. Since in such embodiments, only a portion of the virus that interacts with such a DNA-based nanostructure is covered, encapsulation of one or more viruses or viral particles according to the present invention requires two or more such DNA-based nanostructures to be bound to the one or more viruses or viral particles.

[0052] In the context of the present application, the term "sulfonated or sulfated polysaccharide group" relates to a group comprising a polysaccharide comprising at least one sulfated hydroxyl group or at least one sulfonated glycosylamino group.

[0053] Importantly, in addition to targeting specific receptors, many viruses also weakly interact with different biological substances, including sulfonated or sulfated polysaccharides (Cagno, V. et al., Viruses 11 (2019) 596; see Table 2).

[0054] In particular embodiments, each of the handles comprises two binding sites for the sulfonated or sulfated polysaccharide groups.

[0055] In a particular embodiment, each of the polysaccharides comprising at least one sulfated hydroxyl group or at least one sulfonated glycosylamino group is independently selected from the following list: heparin, heparan sulfate, hybrid heparan sulfate, carrageenan, cellulose sulfate and dextrin 2-sulfate, in particular heparan sulfate or hybrid heparan sulfate.

[0056] In a particular embodiment, the polysaccharide comprising at least one sulfated hydroxyl group or at least one sulfonated glycosylamino group consists of between 3 and 10 disaccharide units, in particular 4, 5, 6, 7, 8 or 9 units, in particular 4 or 9 monosaccharide units.

[0057] In particular embodiments, the disaccharide units comprise two or three O-sulfonate groups and / or N-sulfonate groups, in particular three O-sulfonate groups and / or N-sulfonate groups per disaccharide unit.

[0058] In specific embodiments, each of the sulfonated or sulfated polysaccharide groups is independently selected from the following list: heparin, heparan sulfate, hybrid heparan sulfate, carrageenan, cellulose sulfate, and dextrin 2-sulfate.

[0059] In specific such embodiments, each of the sulfonated or sulfated polysaccharides is independently selected from heparan sulfate and hybrid heparan sulfate, in particular heparan sulfate.

[0060] In the context of the present invention, the terms "heparin" and "heparan sulfate" both refer to a family of linear sulfated heterogeneous polysaccharides found on cell membranes and in the extracellular matrix as part of heparan sulfate proteoglycans (HSPGs). They consist of repeating 1→4 linked disaccharide units, one of which is an α-D-glucosamine residue and the other is a uronic acid (or in salt form, i.e., uronic acid salt). Heparin is a structurally similar polysaccharide found in mast cells as a component of serglycin proteoglycans. Heparan sulfate and heparin can be defined as follows: First, in heparin, the uronic acid salt is mainly α-L-iduronate, while in heparan sulfate, the uronic acid salt is mainly β-D-glucuronate, i.e., the C-5 diastereomer of α-L-iduronate. Second, in heparan sulfate, the D-glucosamine residues are mainly N-acetylated, while in heparin, they are N-sulfonated. Finally, at least 70%-80% of heparin consists of the disaccharide 2-O-sulfated L-iduronate α(1→4)N,6-sulfated D-glucosamine, while in heparan sulfate, approximately 40%-60% of the disaccharide consists of (1→4)D-glucuronate β(1→4)D-glucosamine (which may be N-acetylated or N-sulfonated). Together, these structural features make heparin more sulfated and, therefore, more charged than heparan sulfate. However, it is apparent that the names heparin or heparan sulfate are not as clear as the present description implies, and that polysaccharides isolated from some organisms appear to be hybrid constructs. In the context of the present invention, the term "hybrid heparan sulfate" is used to refer to a hybrid having a structure that is a mixture of "typical" heparin structural elements (L-iduronate; highly sulfonated) and "typical" heparan sulfate structural elements (D-glucuronate; N-acetylated and 6-O-sulfonated).

[0061] Heparan sulfate proteoglycans (HSPGs) (Cagno, V. et al., Viruses 11 (2019) 596; Zhang, Q. et al., Cell Discov. 6 (2020) 1–14) are commonly found on the surface of mammalian cells. Weak interactions between viruses and HSPGs are conserved across virus families and therefore generally appear to be beneficial to the viral life cycle. For example, HSPG-virus interactions can enable infection-enhancing diffusive searches for virus particles to search for specific host cell receptors for virus particles on the surface of cells ( Figure 1A, left). The interaction of heparan sulfate (HS) with viruses has been exploited for medical purposes, such as in virus barrier coatings for condoms based on HS-decorated dendrimers (Tyssen, D. et al., PLOS ONE 5, e12309 (2010); Price, CF et al., PLOS ONE 6, e24095 (2011); Zelikin, AN & Stellacci, F., Adv. Healthc. Mater. 10 (2021) 2001433). Other studies often involve surface functionalization of nanoparticles and polymers with HS derivatives to produce virus-binding complexes with antiviral activity (Cagno, V. et al., Nat. Mater. 17 (2018) 195–203; Al-Mahtab, M. et al., PLOS ONE 11 (2016) e0156667; Vaillant, A., Antiviral Res. 133 (2016) 32–40; Cagno, V. et al., Antimicrob. Agents Chemother. 64 (2020) e02001-20.). Typically, high levels of multivalency are required to increase the binding strength between HS nanoparticles and viruses. The reversible nature of the binding may result in undesirable unbinding and release of infectious viruses from the virus-isolating coating, or the need to maintain high concentrations of therapeutically active agents (Zelikin, loc. cit.).

[0062] In a particular embodiment, the subset of self-assembling DNA-based building blocks consists of between 1% and 100% of all self-assembling DNA-based building blocks that form the DNA-based nanostructure. In a particular embodiment, the subset of self-assembling DNA-based building blocks consists of between 50% and 100%, more particularly between 75% and 100%, and particularly 100% of all self-assembling DNA-based building blocks that form the DNA-based nanostructure.

[0063] In a particular embodiment, one or more of the self-assembling DNA-based building blocks in the subgroup comprises n single-stranded oligonucleotides as the handles, wherein each handle is independently linked to at least one of the sulfonated or sulfated polysaccharide groups, wherein n is an integer independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, in particular wherein n is 9.

[0064] In a particular embodiment, the handle is a single-stranded oligonucleotide having a length between 30 nucleotides and 60 nucleotides, particularly between 40 nucleotides and 55 nucleotides, more particularly between 45 nucleotides and 50 nucleotides.

[0065] Examples of self-assembling DNA structural units in the form of frustum are shown in the examples, wherein the small base of each of the frustums comprises nine of the polynucleotides, such as T-octa self-assembling DNA-based structural units, T1-pentamer-triangle self-assembling DNA-based structural units, T1-ring-triangle self-assembling DNA-based structural units, or T3-6-triangle-type self-assembling DNA-based structural units.

[0066] In a specific embodiment, each member of the n oligonucleotides comprises at least one oligonucleotide fragment as a binding site, and wherein each of the sulfonated or sulfated polysaccharide groups comprises an oligonucleotide having a sequence complementary to one of the oligonucleotide fragments contained in the handle. In a specific embodiment, each member of the n oligonucleotides comprises one or two oligonucleotide fragments as binding sites.

[0067] In certain embodiments, each member of the n oligonucleotides comprises two oligonucleotide fragments as binding sites.

[0068] By using oligonucleotides containing two binding sites, surprisingly, it was possible to demonstrate a further increase in the number of DNA-based nanostructures that actually encapsulated viral particles, even when compared to the already advantageous version with the extended handle design described above. This is particularly surprising in view of the fact that the additional second binding site is located at the same position as the initial binding site in the H1 handle design with the short oligonucleotide (26-mer), and despite the fact that the addition of the second binding site and the HS portion would have been expected to reduce the encapsulation efficiency due to steric hindrance.

[0069] Examples of subsets of nine such oligonucleotide handles can be found in the Hx oligonucleotide groups according to SEQ ID. NOs: 177-185, 186-194, 389-397, 398-406, 607-615, 616-624, 821-829, 1018-1026, 1216-1224, 1416-1424, 1613-1621 and 1812-1820, each oligonucleotide comprising one handle, and the Hx oligonucleotide groups according to SEQ ID. NOs: 195-203, 407-415 and 625-633, each oligonucleotide comprising two binding sites, such oligonucleotide handles can be linked to constructs comprising sulfonated or sulfated polysaccharide groups.

[0070] In particular embodiments, the cavity has a diameter of at least 15 nm, at least 25 nm, at least 50 nm, at least 100 nm, at least 150 nm, at least 200 nm, or at least 250 nm.

[0071] In certain embodiments, the cavity has a diameter of at most 1,000 nm.

[0072] In the context of the present invention, the term "diameter" refers to the diameter of the smallest circle enclosed by the surface of the DNA-based nanostructure. For clarity, in the case of a DNA-based nanostructure in the form of a capsule (or spherocylinder), the diameter is the diameter of the hemispherical end and / or the diameter of the cylindrical center.

[0073] In a specific embodiment, the DNA-based nanostructure has a molecular weight of at least 1 MDa, particularly at least 10 MDa, particularly at least 20 MDa, more particularly at least 30 MDa. In other specific embodiments, the DNA-based nanostructure has a molecular weight of at least 50 MDa, at least 80 MDa, at least 100 MDa, at least 200 MDa, or at least 500 MDa. In a specific embodiment, the DNA-based nanostructure has a molecular weight of up to 1,500 MDa.

[0074] In certain embodiments, the numerical value of the molecular weight of the DNA-based nanostructure (in MDa) is proportional to the numerical value of the volume of the cavity enclosed by the DNA-based nanostructure (in nm). 3 The ratio between the molecular weight and the bounding volume is less than 10,000, in particular less than 9,000. In a particular embodiment, the ratio has a value between 1,000 and 10,000, in particular between 2,000 and 9,000. For example, in the case of certain octahedral nanostructures, wherein the molecular weight is about 40 MDa, and wherein the bounding volume is about 113,000 nm 3 , the ratio is about 2,800.

[0075] In a specific embodiment, the ratio between the surface area of ​​the DNA-based nanostructure covered by the macromolecules forming the DNA-based nanostructure and the surface area not covered by the macromolecules (excluding the opening area of ​​the DNA-based nanostructure in the form of a shell) is at least 1, particularly at least 2, particularly at least 4, particularly at least 6, and particularly at least 8. In other specific embodiments, the ratio is at least 10. In a specific embodiment, the ratio is between 1 and 20, particularly between 2 and 18, between 4 and 16, between 6 and 14, and more particularly between 8 and 12. For example, in the case where the DNA-based nanostructure is a shell in the form of a hemisphere, only the area of ​​the curved surface is used instead of the area of ​​the opening (i.e., the area of ​​the plane of the hemisphere) to calculate the ratio.

[0076] In specific embodiments, the molecular weight of each self-assembling DNA-based building block is between 4.5 MDa and 5.5 MDa.

[0077] In certain embodiments, each self-assembling DNA-based building block comprises between 7,500 and 8,500 base pairs.

[0078] In particular embodiments, the DNA-based nanostructure is composed of between 4 and 180 such self-assembled DNA-based building blocks.

[0079] In certain embodiments, the single-stranded DNA template is single-stranded DNA of a filamentous bacteriophage, or single-stranded DNA derived from a filamentous bacteriophage.

[0080] In the context of the present invention, the term "filamentous phage" refers to a phage or bacterial virus characterized by its filamentous shape, generally containing a circular single-stranded DNA genome and infecting Gram-negative bacteria. Filamentous phages include Ff phages, such as M13, f1 and fd1 phages, and Pf1 phages.

[0081] In certain embodiments, the single-stranded DNA template has the sequence of SEQ ID NO: 1 (M13 8064) (see Table 1). In certain embodiments, the single-stranded DNA is circular.

[0082] In the context of the present invention, a single-stranded DNA template "derived from a single-stranded DNA of a filamentous bacteriophage" refers to a DNA construct derived from a naturally occurring or published filamentous bacteriophage DNA sequence by one or more of the following: (i) opening of a circular structure into a linear sequence; (ii) deleting one or more nucleotides; (iii) inserting one or more nucleotides; (iii) substituting one or more nucleotides; (iv) adding one or more nucleotides; (v) modifying one or more nucleotides. Although any such variation may have a deleterious or at least quite unpredictable effect on the biology of the bacteriophage, its infectivity and its ability to reproduce, such effects do not play any role in the context of the present invention, since, as already mentioned above, the single-stranded DNA template is used only as a naked template, without any requirement for having any functional properties, and all structural aspects, such as the correct formation and three-dimensional shape of the self-assembling DNA-based building blocks, are implemented by appropriately selecting the set of complementary oligonucleotides.

[0083] In a specific embodiment, the single-stranded DNA template has at least 80%, particularly at least 90%, and more particularly at least 95% sequence identity with a naturally occurring or published sequence of a filamentous phage, particularly M13, f1 or fd1 phage, particularly a sequence selected from SEQ ID NO: 1 (M13 8064) and M13 7249 (SEQ ID NO: 2 of WO 2021 / 165528).

[0084] In a particular embodiment, the DNA-based nanostructure is a closed three-dimensional geometric shape, in particular a closed three-dimensional geometric shape selected from the group consisting of a sphere, a spherocylinder and a polyhedron, in particular a tetrahedron, an octahedron or an icosahedron, which is formed in situ from said self-assembling DNA-based building blocks in the presence of said virus or viral particle to be encapsulated.

[0085] In specific such embodiments, the self-assembling DNA-based building blocks that form the DNA-based nanostructure in situ are a collection of individual DNA-based building blocks, each of which comprises a single single-stranded DNA template.

[0086] In specific other embodiments, the self-assembling DNA-based building block that forms the DNA-based nanostructure in situ is a collection of one or more pre-assembled DNA-based building blocks composed of two or more individual DNA-based building blocks, each building block comprising a single single-stranded DNA template.

[0087] In certain embodiments, all of the self-assembling DNA-based building blocks are pre-assembled DNA-based building blocks. In an alternative embodiment, the self-assembling DNA-based building blocks are a mixture of pre-assembled DNA-based building blocks and separate DNA-based building blocks, each building block comprising a single single-stranded DNA template.

[0088] In such an embodiment, the preassembled DNA-based building blocks form a curved geometry, wherein the handle or the construct comprising at least one sulfonated or sulfated polysaccharide group attached to the handle is present on the negative curvature of the curved geometry such that the handle or construct points toward the interior of the cavity formed by self-assembly of the preassembled DNA-based building blocks.

[0089] In another specific embodiment, the DNA-based nanostructure is a shell having an opening for accessing the cavity.

[0090] In the context of the present invention, the term "shell" refers to a structure that is part of a closed three-dimensional geometric shape, in particular a closed three-dimensional geometric shape selected from a sphere, a spherocylinder and a polyhedron, in particular a tetrahedron or an octahedron.

[0091] In yet another specific embodiment, the DNA-based nanostructure is a combination of a first subshell and a second subshell, each of the first subshell and the second subshell having an opening into a first lumen and a second lumen, respectively, wherein the first lumen and the second lumen together form the cavity.

[0092] In a specific embodiment, the first subhousing and the second subhousing are connected by at least one joint.

[0093] In certain embodiments, the linker is a linker selected from the group consisting of a DNA linker, an RNA linker, a polypeptide linker, a protein linker, and a chemical linker.

[0094] In the context of the present invention, the term "DNA linker" refers to a linker formed by DNA, wherein the sequence of the DNA linker is not complementary to the DNA of the single-stranded DNA template or to any of the oligonucleotide sets complementary to the single-stranded DNA template, wherein the DNA linker is linked at one end to a DNA sequence of a self-assembling DNA-based structural unit forming the first shell, and at the other end to a DNA sequence of a self-assembling DNA-based structural unit forming the second shell.

[0095] In the context of the present invention, the term "polypeptide linker" refers to a linker formed by at least 2, in particular at least 5, at least 10 or at least 20 amino acid residues connected by peptide bonds, wherein the polypeptide has no tertiary or quaternary structure, and wherein the polypeptide linker is linked at one end to a DNA sequence forming a self-assembling DNA-based building block of the first shell, and at the other end to a DNA sequence forming a self-assembling DNA-based building block of the second shell.

[0096] In the context of the present invention, the term "protein linker" refers to a linker formed by at least 20, in particular at least 50, at least 100, at least 200 amino acid residues, at least 500 amino acid residues or at least 1000 amino acid residues (in particular less than 1,500 amino acid residues) connected by peptide bonds, wherein the polypeptide has a tertiary and / or quaternary structure, and wherein the protein linker is connected at one end to the DNA sequence of the self-assembling DNA-based structural unit forming the first shell, and at the other end to the DNA sequence of the self-assembling DNA-based structural unit forming the second shell. In a specific embodiment, the protein linker is covalently linked to the DNA sequence. In specific other embodiments, the protein linker is non-covalently linked to the DNA sequence, in particular, wherein the protein linker is an antibody-based protein linker, in particular selected from diabodies and full antibodies, including IgG antibodies.

[0097] In the context of the present invention, the term "chemical linker" refers to a continuous chain of between 1 and 30 atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 atoms); therefore, in the context of the present invention, the term "between" is used so that the mentioned boundaries are included in its backbone, i.e. the length of the linker is defined as the shortest connection as measured by the number of atoms or bonds between the two DNA sequences connected by the chemical linker. In the context of the present invention, a chemical linker is preferably a C 1-20 -alkylene, C 1-20 -heteroalkylene, C 2-20 -Alkenylene, C 2-20 -heteroalkenylene, C 2-20 -Alkynylidene, C 2-20-heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene or heteroaralkylene groups, which may be optionally substituted. The joint may contain one or more structural elements, such as formamide, ester, ether, thioether, disulfide, urea, thiourea, hydrocarbon moiety, etc. The joint may also contain a combination of two or more of these structural elements. Each of these structural elements may exist in the joint more than once, for example twice, three times, four times, five times or six times. In certain embodiments, the joint may include a disulfide bond. It should be understood that the joint must be connected to the two DNA sequences connected by the chemical joint in a single step or in two or more subsequent steps. For this reason, the standby joint will carry two groups, preferably at the proximal and distal ends, which may (i) form a covalent bond with a group present in one of the two DNA sequences to be connected, or (ii) they may be activated or may be activated to form a covalent bond with one of the two DNA sequences.

[0098] In certain embodiments, the DNA-based nanostructure is based on an icosahedral structure.

[0099] In particular embodiments, each of the self-assembling DNA-based building blocks is a prism.

[0100] In the context of the present invention, the term "prism" refers to a polyhedron in which all vertices lie in two parallel planes.

[0101] In certain embodiments, the prism is a triangular prism. In other embodiments, the prism is a rectangular prism.

[0102] In certain embodiments, the DNA-based nanostructures are based on a mixture of triangular prisms and rectangular prisms.

[0103] In certain embodiments, the present invention relates to DNA-based nanostructures.

[0104] Each of the triangular prisms or rectangular prisms is formed by m triangular planes or rectangular planes, respectively.

[0105] wherein m is an integer independently selected from 4, 5, 6, 7 and 8, particularly an integer independently selected from 5, 6 and 7, more particularly wherein the integer is 6.

[0106] wherein three sides or four sides of each of the m planes are respectively formed by n parallel DNA double helix segments, wherein n is an integer independently selected from 1, 2, 3, 4, 5 and 6, particularly an integer independently selected from 2, 3, 4 and 5, more particularly an integer independently selected from 3 and 4, wherein each plane is connected to a plane above the plane and / or a plane outside the plane (i) through stacking interactions between the DNA double helices forming the planes and (ii) in part through DNA segments within the single-stranded DNA template and / or the oligonucleotide forming the DNA-based building unit bridging at least two of the planes, and wherein at least two of the three or four side trapezoids respectively contain a specific pattern of recesses and / or protrusions formed by missing or additional DNA double helix segments for specific interaction with a complementary pattern on the side trapezoid of another one of the self-assembling DNA-based building units.

[0107] In specific embodiments, the average length of each of the n DNA double helix segments in the m planes of the triangular prism or rectangular prism, respectively, is between 80 and 200 base pairs.

[0108] In a particular embodiment, the triangular prism is a triangular frustum. In a particular embodiment, the rectangular prism is a rectangular frustum.

[0109] In the context of the present invention, the term "triangular frustum" refers to a three-dimensional geometric shape in the form of a triangular cone, and the term "rectangular frustum" refers to a three-dimensional geometric shape in the form of a rectangular cone, wherein the tip of the cone has been removed, thereby creating a plane at the top that is parallel to the bottom of the cone.

[0110] In a specific embodiment, for at least a portion of the self-assembling DNA-based building block, the length of at least one side of each of the m planes gradually decreases from the first plane to the m-th plane, so that an oblique angle is generated between a plane perpendicular to the first plane and a trapezoidal plane formed by the m sides (see WO 2021 / 165528). Figure 5 ). In certain embodiments, all three or four trapezoidal planes are each at an oblique angle.

[0111] In a particular embodiment, the bevel angle is between 16° and 26°, particularly between 18° and 24°, more particularly between 20° and 22°, most particularly about 20.9°.

[0112] In certain embodiments, the DNA-based nanostructure comprises at least one set of self-assembling DNA-based structural units, wherein all three or four side ladders respectively comprise a specific pattern of recesses and / or protrusions formed by missing or additional DNA double helix segments, which is used to specifically interact with a complementary pattern on the side ladder of another of the self-assembling DNA-based structural units.

[0113] In certain embodiments, particularly where the DNA-based nanostructure is a closed three-dimensional geometry, all of the self-assembling DNA-based building blocks are identical.

[0114] In certain embodiments, the DNA-based nanostructure comprises two or more sets of self-assembling DNA-based building blocks.

[0115] In certain embodiments, the DNA-based nanostructures are rod-shaped.

[0116] In certain embodiments, the DNA-based nanostructure comprises two or more sets of self-assembling DNA-based building blocks.

[0117] In specific such embodiments, the rod-shaped DNA-based nanostructures comprise at least a first and a second group of self-assembling DNA-based building blocks, wherein the first and second groups differ at least in bevel angles. In a particular embodiment, at least one group consists of self-assembling DNA-based building blocks that exhibit only two bevel angles. In a particular embodiment, the at least one group consists of rectangular frustums that include a bevel angle on each of two opposing trapezoids.

[0118] In certain embodiments, the side trapezoids forming the sides of the shell or the sides of the first and second shells, respectively, do not include a specific pattern of recesses and / or protrusions formed by missing or extra DNA double helical segments, which is used to specifically interact with a complementary pattern on the side trapezoid of another of the self-assembling DNA-based building blocks.

[0119] In certain embodiments, the DNA-based nanostructure is a shell selected from:

[0120] (i) Semi-octahedral T_octa (WO 2021 / 165528 Figure 4 A), which consists of a set of four copies of a triangular frustum in which the base pair stacking contacts on one of the triangular sides of the triangular frustum are inactivated by chain shortening or by the addition of unpaired thymidine (WO 2021 / 165528 Figure 4 A,

[0121] See Figures 24A, D of WO 2021 / 165528);

[0122] (ii) T = 1 half shell (WO 2021 / 165528 Figure 4 B), which consists of two groups of five copies of two different triangular frustums in each case, wherein the five copies of the first group form a closed pentamer and the five copies of the second group are docked to the edges of the pentamer (WO 2021 / 165528 Figure 4 B, see Figures 24B, E of WO 2021 / 165528); and

[0123] (iii) "Trap-like" T=1 shell with missing pentagonal vertices (WO 2021 / 165528 Figure 4 C), which consists of three groups of five copies in each case of three different triangular frustums, wherein the five copies of the first group form a closed pentamer, the five copies of the second group are docked to the edge of the pentamer, the five copies of the third group are docked to the edge of the pentamer, and the five copies of the third group are docked into the gaps between the five copies of the second group (WO2021 / 165528 Figure 4 C, see WO

[0124] Figure 24C, F of 2021 / 165528);

[0125] (iv) T=3 icosahedral half-shells consisting of a total of 30 triangular subunits divided into five copies of six different full-size DNA triangle designs with specific edge docking rules

[0126] In a specific embodiment, the present invention relates to a DNA-based nanostructure, which further comprises one or more types of DNA brick constructs, each type of such DNA brick constructs being characterized by one or more interaction sites for specifically interacting with one or more complementary interaction sites on the planes of the triangular prism or rectangular prism, respectively, present on the outer surface of the DNA-based nanostructure through edge-to-edge stacking contact, wherein the DNA brick construct covers the free space between three or four sides of the plane, respectively (see Figure 33 of WO 2021 / 165528).

[0127] In certain embodiments, the present invention relates to a DNA-based nanostructure further comprising one or more cross-links within one of the triangular prisms or rectangular prisms, respectively, and / or between both of the triangular prisms or rectangular prisms, respectively.

[0128] In the context of the present invention, the term "crosslinking portion" refers to any permanent or intermittent connection within one of the triangular prisms or rectangular prisms and / or between two of the triangular prisms or rectangular prisms, respectively. Any such connection can be pre-realized by connecting two oligonucleotides used to form the self-assembling DNA-based building block before assembly, or for example by adding connections chemically or photochemically between different parts of the three-dimensional nanostructure. Permanent connections can be produced, for example, by appropriately photochemically crosslinking T residues located in the structure that has formed covalent cyclobutane pyrimidine dimer (CPD) bonds (41), and intermittent connections can be produced, for example, by photochemically crosslinking between 3-cyanovinylcarbazole (cnvK) moieties (located at the first blunt end) and thymine residues (T) (located at the other blunt end) at the blunt ends of the two double helical subunits (40).

[0129] In a second aspect, the present invention relates to a composition comprising a DNA-based nanostructure according to the present invention encapsulating one or more viruses or viral particles.

[0130] In certain embodiments, the composition is formed during the process of removing the virus or viral particle from a medium containing the virus or viral particle. In certain other embodiments, the composition is formed during the process of incorporating the one or more viruses or viral particles as cargo into the DNA-based nanostructure.

[0131] In a third aspect, the present invention relates to a method for encapsulating one or more viruses or viral particles, comprising the steps of providing a DNA-based nanostructure according to the present invention, and contacting the DNA-based nanostructure with a medium containing or suspected of containing the virus or viral particles.

[0132] In certain embodiments, (i) for a size of at most 50*50*50 nm 3 For viruses, DNA-based semi-shell nanostructures based on T_octa self-assembling DNA-based building blocks were selected; (ii) for viruses with sizes between 15*15*15 and 100*100*100nm 3 For viruses between 15*15*15 and 100*100*100 nm, a DNA-based semi-shell nanostructure based on a T1_pentamer_triangular self-assembling DNA-based structural unit was selected; (iii) for viruses with a size between 15*15*15 and 100*100*100 nm, a DNA-based semi-shell nanostructure based on a T1_pentamer_triangular self-assembling DNA-based structural unit was selected 3 For viruses between 50 and 50 nm, a DNA-based semi-shell nanostructure is selected based on a combination of a T1-pentamer-triangle self-assembly type DNA-based structural unit and a T1-ring-triangle self-assembly type DNA-based structural unit; and / or (iv) for a virus with a size of 50*50*50 nm3 For viruses larger than 100 μg / ml, DNA-based half-shell nanostructures based on T3_6_triangle self-assembling DNA-based building blocks were selected.

[0133] In certain embodiments, the method is used to remove the one or more viruses or viral particles from the medium. In certain embodiments, the method is used to encapsulate the one or more viruses or viral particles for transporting the viruses or viral particles.

[0134] In a fourth aspect, the present invention relates to a method for encapsulating one or more viruses or viral particles, comprising the steps of providing a DNA-based nanostructure according to the present invention, and contacting the DNA-based nanostructure with a medium containing or suspected of containing the virus or viral particles.

[0135] In an alternative aspect, the present disclosure provides a method for encapsulating one or more viruses or viral particles, comprising the following steps: adding a self-assembling DNA-based structural unit to a medium containing or suspected of containing the viruses or viral particles, thereby forming in situ a DNA-based nanostructure according to the present invention that encapsulates one or more of the viruses or viral particles.

[0136] In yet another aspect, the present disclosure provides a method for encapsulating cargo other than viruses or viral particles (such as complex macromolecules), comprising the steps of providing a DNA-based nanostructure according to the present invention, and contacting the DNA-based nanostructure with a medium containing or suspected of containing the cargo.

[0137] In an alternative aspect, the present disclosure provides a method for encapsulating cargo other than viruses or viral particles (such as complex macromolecules), comprising the following steps: adding self-assembling DNA-based structural units to a medium containing or suspected of containing the cargo, wherein the cargo is other than viruses or viral particles, such as complex macromolecules, thereby forming in situ a DNA-based nanostructure according to the present invention that encapsulates the cargo other than viruses or viral particles (such as complex macromolecules).

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[0200] It should be understood that certain features of the invention described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, different features of the invention described in the context of a single embodiment for the sake of brevity may also be provided separately or in any suitable sub-combination. All combinations of embodiments of the invention are specifically included in the present invention and are disclosed herein as if each and every combination were individually and clearly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically included in the present invention and disclosed herein as if each and every such sub-combination were individually and clearly disclosed herein.

[0201] The present invention is not limited to the scope of the specific embodiments described herein. In fact, various modifications of the present invention other than the modifications described herein will become apparent to those skilled in the art based on the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0202] To the extent permitted by applicable patent law, all patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference.

[0203] The following examples illustrate the invention described above, however, are not intended to limit the scope of the invention in any way.Other test models also known to those skilled in the art can also determine the beneficial effects of the claimed invention.

[0204] Examples

[0205] introduce

[0206] Effective broadband antiviral platforms that can act on existing and yet-to-be-emerged viruses are not yet available, and therapeutic strategies that go beyond conventional approaches need to be explored. Here, we report viral encapsulated DNA origami capsids that achieve broadband viral capture properties by exploiting the broad background affinity of viruses for heparan sulfate proteoglycans (HSPGs). By carefully shaping the interior of the DNA origami capsids with calibrated heparan sulfate (HS) derivative densities, we can successfully encapsulate adenovirus, adeno-associated virus, chikungunya virus, dengue virus, human papillomavirus, norovirus, poliovirus, rubella virus, and SARS-CoV-2 virus or virus-like particles in the same HS-functionalized capsid system without the need for virus type-specific binders. Our HS-functionalized capsids amplify the individual weak and reversible interactions of HSPGs with the viral surface through the strong avidity effect that occurs when the curvature-matched HS-coated capsid engulfs the viral particle. Depending on the relative size of the capsid and the viral particle, it is also possible for each capsid to capture multiple viral particles, and multiple capsids can also coordinate and surround clusters containing dozens of viral particles. Since steric closure in viral endosomes can prevent virus from interacting with host cells, heparan sulfate-coated viral endosomes open up an attractive avenue for establishing broad-spectrum antiviral therapeutic strategies.

[0207] Example 1: Shell design and synthesis principles

[0208] Here, we addressed the challenge of developing broad-spectrum antiviral drugs by exploiting the conserved context of HSPG binding to viruses to irreversibly trap the virus in HS-functionalized neutralizing capsids ( Figure 1 A, right).

[0209] To capture viruses of different sizes, we fabricated three DNA origami shell variants and functionalized their interiors with the same HS derivatives. We used the previously described octahedral and T=1 icosahedral half-shell designs (O and T1, respectively; Sigl et al., loc. cit.), which involved cavities with widths of 40 nm and 85 nm, respectively ( Figure 1 D). We also developed a new T=3 icosahedral half-shell design, called T3, for encapsulating larger viral particles that cannot fit into the O and T1 shells ( Figure 1 E). The T3 design is a finite-size higher-order assembly consisting of a total of 30 triangular subunits divided into five copies of six different full-size DNA origami triangle designs with specific edge docking rules ( Figure 6 The resulting shell has a cavity diameter of approximately 150 nm. Negative staining transmission electron microscopy (TEM) images confirmed the successful assembly of the T3 shell ( Figure 1 F and Figure 7 ).

[0210] Unless otherwise stated, staple strands for origami folding reactions were purchased from Integrated DNA Technologies (IDT) and used by standard desalting purification. DBCO-modified handle strands were purchased from Biomers, HPLC grade. Azide-modified heparan sulfate derivatives were purchased from Glycan Therapeutics (catalog references: 1a: GT24-AZ-021; 1b: GT24-AZ-005; 1c: GT18-AZ-003; 1d: custom products). VLPs were purchased from The Native Antigen Company, Creative Biostructure, and Creative Biolabs (catalog references can be found in Table 12).

[0211] Table 12: VLP suppliers and catalog references

[0212] VLP supplier refer to Poliovirus type 3 Creative Biolabs VLP-003YF Dengue virus type 1 The Native Antigen Company DENV1-VLP-100 G II.4 Norovirus The Native Antigen Company REC31620-100 HPV 16 Creative Biostructure CBS-V641 HPV16 Chikungunya virus The Native Antigen Company CHIKV-VLP-10 SARS-CoV-2 Creative Biolabs VLP-050YF Rubella virus The Native Antigen Company REC31651-100

[0213] Folding of DNA origami triangle subunits

[0214] DNA origami structures were folded in a one-pot reaction containing 50 nM single-stranded scaffold DNA (M13, 8064 bases) and 250 nM of each staple chain in a standard "folding buffer" (FoBx) (pH 8.00) containing x = 20 mM MgCl2, 5 mM Tris base, 1 mM EDTA and 5 mM NaCl. As previously described, scaffold M13 (SEQ ID NO: 1; Engelhardt, FAS et al., ACS Nano 13 (2019) 5015–5027) was generated based on M13 8064 as a scaffold sequence. All folding reactions were subjected to optimized thermal annealing ramps (Table 13) in a Tetrad (Bio-Rad) thermal cycler. It should be noted at this point that any variant of M13 8064 or in fact any other single-stranded DNA of sufficient length can be used as a scaffold sequence with a set of staple chains of corresponding design. Alternatively, DNA origami structures of the type used in this application can be constructed by using different sets of overlapping single-stranded oligonucleotides and standard DNA origami techniques. Table 13: Temperature ramps and scaffolds for each DNA origami triangle subunit. For scaffold sequences, see SEQ ID NO: 1 in Table 1. For staple sequences, see Tables 2 to 10.

[0215]

[0216] Purification of triangular subunits and capsid self-assembly

[0217] All origami structures were purified using agarose gel extraction (1.5% agarose containing 0.5× TBE and 5.5 mM MgCl2) and centrifuged at maximum speed for 30 min to precipitate the residual agarose. If the origami required a concentration step, ultrafiltration (Amicon Ultra 500 μl, 100 kDa molecular weight cutoff) was performed before shell assembly. For shell assembly, the purified triangles were mixed in a 1:1 ratio. Typical triangle subunit concentrations ranged from 5 nM to 400 nM, while assembly times depended on the shell type. Table 14 summarizes and provides a comparison of the optimized salt concentrations, temperatures, and self-assembly times required for all shells used in this study.

[0218] Table 14: Half-shell assembly conditions.

[0219] structure <]]> Temperature(℃) time O Shell 40 40 overnight T1 housing 40 40 24h T3 Housing 25 40 8 weeks

[0220] The assembled shells were UV cross-linked at 310 nm using an Asahi Spectra xenon light source 300W MAX-303 for 1 h (Gerling, T. et al., Sci. Adv. 4 (2018) eaau1157). Prior to VLP encapsulation experiments, buffer exchange with 1× PBS containing 10 mM MgCl was performed using ultrafiltration (Amicon Ultra 500 μl, molecular weight cutoff 100 kDa) or dialysis (D-TubeTM microdialyzer, MWCO 12–14 kDa, 2×500 ml, 8 h exchange, room temperature).

[0221] Ligation of heparan sulfate to DNA

[0222] We used strain-promoted azide-alkyne 1,3-dipolar cycloaddition (SPAAC) to covalently attach heparan sulfate derivatives to DNA oligonucleotides ( Figure 1 B. Figure 5), which can hybridize to specific receptor sites on the interior of the DNA origami shell: single-stranded DNA stretches called “handles”. For conjugation, we used HS derivatives modified with azide groups (1a and 1c, respectively) containing 8 or 18 sugar monomers (including monomers that characterize HS polymers, such as N-acetyl-glucosamine and glucuronic acid). As controls, we used 8-mer and 18-mer polysaccharides lacking sulfate and sulfonate groups (1b and 1d, respectively). The DNA oligonucleotides to be clicked to the different HS polymers were modified with a dibenzocyclooctyne (DBCO) moiety (2), and the SPAAC reaction occurred rapidly after the two components were mixed. We analyzed the reaction products by polyacrylamide gel electrophoresis (PAGE) (3a-d), revealing different electrophoretic mobilities of the different product versions, consistent with expectations ( Figure 1 C). Higher molecular weight reaction products have slower mobilities, and products containing sulfate groups migrate faster in the gel compared to products lacking sulfate groups, which we attribute to the additional negative charge.

[0223] Excess azide-modified heparan sulfate derivatives (1a-d) were mixed with DBCO-modified DNA in a ratio of 4:1 to form the corresponding products (3a-d). MgCl2 was added to a concentration of 0.5M and the mixture was placed at 37°C overnight to achieve a conversion rate of >90%. The product was run at 35W for 2h in a preparative 10% PAGE gel. Subsequently, the product band was cut and crushed. 1×TEN buffer (10mM Tris-HCl, 1mM EDTA, 100mM NaCl, pH 8.00) was added to dissolve and recover the modified oligonucleotides, and concentrated and buffer exchanged using EtOH precipitation. The pure product was redissolved and stored in twice distilled H2O at 4°C or -20°C.

[0224] Attachment of heparan sulfate-modified DNA constructs to DNA origami capsids

[0225] We then hybridized HS-modified DNA oligonucleotides to single-stranded DNA handles complementary in sequence to those protruding from the inner surface of the target DNA origami shell.

[0226] Testing different heparan sulfate-conjugated DNA origami capsids

[0227] In preliminary experiments using adeno-associated virus serotype 2 (AAV2), we explored three different DNA handle designs: proximal (H1), distal (H2), and branched (H3) to determine the type and density of HS modifications required for efficient viral capture ( Figure 1G). These initial experiments using the O half-shells showed that H1 was the least efficient, and both the H1 and H2 designs were less efficient for viral capture than the H3 branch handle design. Samples were analyzed via negative stain transmission electron microscopy (TEM), where images were collected using an automated montage setting to minimize bias. Blind TEM quantification of particles revealed that when H3 was hybridized with the 18-mer HS derivative (3c), approximately 96% of the shells were occupied by AAV2, which is an improvement over the approximately 30% and 84% occupied shells achieved with H1 and H2, respectively ( Figure 8 ). Therefore, we henceforth used branch handles to design H3 and HS 18-mer variants (3c), unless otherwise stated. We confirmed that the interaction with AAV2 was due to the sulfate and sulfonate groups present in the HS structure, since the 3d HS derivative used as a negative control did not show any binding ( Fig. 9 Importantly, all AAV2 particles were captured by excess O capsids ( Fig.10 ).

[0228] Example 2: Capturing different viruses using DNA origami capsids

[0229] Using the HS handle design thus established, we tested the ability of HS-modified DNA origami shells to capture a variety of exemplary viruses and virus-like particles (VLPs) (Zeltins, A., Mol. Biotechnol. 53 (2013) 92–107). Our target virus library sampled enveloped and non-enveloped particles, particles from different virus families, and particles with a size range of 25 to 90 nm (Table 11, see also Fig.11 TEM image of the PDMS chip).

[0230] Maturation of dengue virus VLPs

[0231] Dengue virus VLP maturation was regulated by published methods (Yu, I.-M. et al., Science 319 (2008) 1834-1837; Yu, I.-M. et al.; J. Virol. 83 (2009) 12101-12107). Briefly, dengue virus VLP samples (10 μl, 0.39 mg / ml, The Native Antigen Company, catalog number DENV1-VLP) were added to MES buffer (10 μl, 50 mM, pH 6.00) and gently mixed. Next, CaCl was added. 2(水溶液)(0.75 μl 0.1 M) and furin (3.9 μl, 2000 U / ml, New England Biolabs, catalog number P8077) were added and mixed, and the samples were incubated at 30°C for 16 h. After incubation, Tris buffer (25 μl 100 mM Tris-HCl, 120 mM NaCl, pH 8.00) was added to the samples, and the samples were immediately dialyzed against 1× PBS (D-Tube TM The samples were dialyzed using a Dialyzer Mini, MWCO 12–14 kDa, 2 × 50 ml exchanges over 24 h, 4°C). The mature dengue virus VLP samples were used immediately and stored at 4°C.

[0232] Virus and VLP encapsulation

[0233] We used HS-modified O capsids to sequester AAV2, poliovirus, mature dengue virus, and norovirus ( Figure 2 a); Use of HS-modified T1 capsids to capture human papillomavirus type 16 (HPV 16), SARS-CoV-2, chikungunya virus, and rubella virus particles ( Figure 2 b); and using HS-modified T3 capsid to enclose adenovirus type 5 (TEM tomography) Figure 2 c and Fig.12 ).

[0234] Preassembled and UV-welded shells in 1× PBS containing 10 mM MgCl2 were mixed with VLP samples in appropriate ratios to achieve an excess of shells or VLPs. The MgCl2 concentration was adjusted to 10 mM and the samples were incubated at room temperature for 2 h. The usual amount of sample for TEM analysis ranged from 5 μl-10 μl total solution, with a triangle origami concentration of approximately 10 nM triangle origami concentration. Negatively stained TEM grids were prepared immediately after the 2 h incubation.

[0235] Negative stain TEM

[0236] The samples were incubated on a Cu400 TEM grid (Electron Microscopy Sciences) coated with formvar carbon by glow discharge (45s, 35mA) for 90s to 120s, depending on the origami and MgCl2 concentration. Next, the grid was stained for 30s with a 2% aqueous uranyl formate solution containing 25mM NaOH. Imaging was performed at magnifications between 10,000 and 42,000 in a SerialEM in a FEI TecnaiT12 microscope, operated at 120kV, with a Tietz TEMCAM-F416 camera. TEM micrographs were high-pass filtered to remove long-range staining gradients, and contrast was automatically leveled using Adobe Photoshop CS5. In order to obtain TEM statistics in an unbiased manner, automatic grid montages were obtained. In order to obtain detailed information on the selected particles, negative staining EM tomography was used as a visualization technique. Tilt series were performed from -50° to +50°, and micrographs were acquired in 2° increments.

[0237] Tilt series were processed with Etomo (IMOD) to obtain tomograms (Kremer, J. et al., J. Struct. Biol. 116 (1996) 71-76). Micrographs were aligned to each other by calculating the cross-correlation of consecutive tilt series images. Tomograms were then generated using filtered back projection. A Gaussian filter was used with a cutoff value between 0.25 and 0.5 and an attenuation value of 0.035.

[0238]

[0239] Interestingly, in many cases, the multivalent interactions between the HS coating on the interior of the capsid and the viral particle appear to be strong enough to support large elastic deformations of the surrounding capsid. For example, the T3 capsid material deforms from a spherical to an ellipsoidal shape around an adenovirus particle, which may be driven by maximizing the number of molecular interactions between the HS moieties on the interior surface of the capsid and the viral surface, but at the expense of elastic deformation of the capsid. The O capsid occasionally deforms so that up to four AAV2 particles can fit in its cavity ( Figure 3 a), although by design, if the O capsid were completely rigid, it would only have room for one AAV2 particle. The T1 capsid can also bend to accommodate up to three copies of HPV 16 ( Figure 3 b). Depending on the relative stoichiometry between capsid and virion, we also observed sandwich-like structures in which two capsids coordinate one virion (e.g., HPV 16 and O capsids, Figure 3c). If the capsid diameter greatly exceeds the target virus size, multiple target particles can be sequestered. For example, we observed up to six AAV2 ( Figure 3 d), and up to three Chikungunya viruses were observed in the T3 capsid (TEM tomography Figure 3 e and Fig.13 In addition, multiple copies of HS-modified capsids can also cooperatively encapsulate dozens of AAV2 particles in clusters surrounded and protected by the DNA origami capsid material ( Figure 3 f) These results support the view that the capsid is able to flexibly adapt and capture more pleomorphic virus particles.

[0240] In the negative stain TEM images, we see that the Chikungunya virus VLP particles appear to completely fill the T1 cavity. Presumably due to the resulting high degree of shape complementarity, we can use Figure 1 Any of the different handle designs described in G effectively captured Chikungunya virus particles within T1 capsids with high yields (H1, 3a, 90% full capsids). In fact, even using the 3b negative control (which is a capsid with a coating lacking sulfate and sulfonate groups), we could capture Chikungunya virus, but with lower yields (H1, 3b, 54% full capsids, see also Fig.14 ). We interpret this phenomenon as a manifestation of molecular recognition at the mesoscopic scale. This effect may be due to the synergistic amplification of weak electrostatic interactions between the negatively charged DNA capsid and the chikungunya virus particle as they interact over an enlarged surface area. Incidentally, this observation also suggests an alternative modification-free virus capture pathway that considers precisely tailoring the capsid to the size of the target virus.

[0241] Dengue virus and some other viruses present two different "mature" and "immature" conformations. Viral surface proteins must undergo certain conformational changes to become infectious, allowing them to move between carriers and hosts and / or infected cells and healthy cells (Yu, I.-M. et al., Science 319 (2008) 1834-1837; Yu, I.-M. et al., J. Virol. 83 (2009) 12101-12107; Lim, X.-X. et al., Nat. Commun. 8 (2017) 14339; San Martín, C., Virus Maturation. See: Physical Virology: Virus Structure and Mechanics (Greber, UF ed.) 129-158 (Springer International Publishing, 2019), doi: 10.1007 / 978-3-030-14741-9_7). Although VLPs are very convenient to use for safety reasons, we acknowledge that there are some limitations. For example, initially our dengue virus VLP samples contained a high percentage of immature particles that did not bind to our HS-functionalized capsids. To overcome this problem, we induced enzymatic maturation of dengue virus VLPs, as occurs in vivo, and observed binding of mature particles ( Figure 2 aDengue virus and Fig.15 ).

[0242] We also performed cryogenic electron microscopy (cryo-EM) measurements of HPV 16 and Chikungunya virus VLPs captured within O and T1 capsids, respectively ( Figure 4 ).

[0243] Cryo-EM

[0244] DNA origami shells were prepared and functionalized and viruses were captured as described above. The samples (O+HPV: 70nM triangles; T1+Chikungunya virus: 200nM triangles) were incubated on a glow-discharged lace carbon 400 mesh copper grid with an ultrathin carbon film for 60s. Subsequently, the grids were rapidly frozen in liquid ethane using a FEI Vitrobot Mark V (imprint time: 2.5s, imprint force: -1, discharge time: 0s, 22°C, 100% humidity, 3μl sample). Cryo-EM imaging was performed using a spherical aberration (Cs) corrected TitanKrios G2 electron microscope (Thermo Fisher), which was operated at 300kV and equipped with a Falcon III 4k direct electron detector (Thermo Fisher). Automated image acquisition was performed in EPU 2.6 (dose: Exposure time: 3s-5s, 12 fractions, pixel size: 0.23nm (O+HPV) and 0.29nm (T1+Chikungunya virus), defocus: -1.5μm to -2μm). Micrographs were processed in RELION-3 (Zivanov, J. et al., eLife 7 (2018) e42166) using MotionCor2 (Zheng, SQ et al., Nat. Methods 14 (2017) 331–332) and CTFFIND4.1 (Rohou, A. and Grigorieff, N., J. Struct. Biol. 192 (2015) 216–221). Particles were automatically picked using cryYOLO 1.7.6 (Wagner, T. et al., Commun. Biol. 2 (2019) 1–13). The extracted particle images were classified and selected by visual inspection through multiple rounds of 2D and 3D classification. The initial model was generated in silico in RELION-3. 3D reconstruction and multibody refinement produced an electron density map with a resolution of 1.3 for the O capsid of the captured HPV. (EMD-13884, 1×O+HPV: 7834 particles, 2×O+HPV: 4634 particles), and for capturing the T1 capsid of Chikungunya virus, the resolution was (EMD-13883, 1259 particles, C5 symmetry).

[0245] Two-dimensional (2D) class-averaged images and 3D cryo-EM reconstructions confirmed that the VLPs were successfully trapped within the cavities of the corresponding shells ( Figure 4 b, Figure e and Figure 16 to Figure 17 Although one O shell is not large enough to encapsulate the entire HPV 16 particle, two O copies can coordinate and completely cover the entire VLP ( Figure 4c). 2D class averages of free HPV 16 showing size variation within the VLP samples ( Fig.18 ). Consistently, we also found that the gap distance between O shells (e.g. Figure 4 The white arrow in b) varies depending on whether a smaller or larger HPV 16 particle is captured. The cryo-EM image of the complex we determined consisting of Chikungunya virus VLPs in a HS-modified T1 capsid revealed a near-perfect fit between the two particles ( Figure 4 e, f). The cryo-EM images convincingly illustrate the relative size range of artificial DNA origami capsids relative to their viral targets, and the extent of surface confinement that can be achieved by sequestering the virus within the capsid.

[0246] Example 3: Stability of DNA origami capsids encapsulating viruses / VLPs

[0247] Finally, to test the stability of capsid-captured virus, we serially diluted an exemplary sample consisting of AAV2 encapsidated in O capsids. Relative to the undiluted sample, the fraction of occupied capsids remained the same in the diluted samples before and after a 100-fold dilution and 14 days of incubation ( Fig.19 ), indicating that the spontaneous dissociation rate of the complex formed between the AAV2 particle and the surrounding HS-modified capsid is at least in the range of weeks under the conditions tested. The high stability is driven by avidity and can be understood by considering that spontaneous dissociation of AAV2 from the surrounding capsid requires the simultaneous disruption of dozens of bonds formed between the HS chains on the endosome and the viral surface. The probability of such an event decreases exponentially with the number of HS bonds formed.

[0248] Example 4: Efficiency of DNA origami capsid encapsulation of viruses / VLPs

[0249] 1-Handle design and development for effective virus capture

[0250] To optimize and calibrate the required density for our heparan sulfate derivatives, we used three different handle variants ( Figure 1 G and Figure 8 a) The capture efficiency of O halfshells for AAV2 was explored exemplarily. The proximal handle (H1) was the shortest design tested and consisted of a 26-nucleotide DNA stretch, positioning the heparan sulfate modification in the proximal arrangement. The distal handle design (H2) included a single-stranded extension of 20 thymidines (polyT extension), allowing the heparan sulfate groups to reach farther from the origami surface and increase the chance of multivalent binding events. Finally, the branched design (H3) mimicked a branched polymer with two heparan sulfate modifications per handle unit, doubling the local heparan sulfate density.

[0251] The three handle designs were tested in parallel using O half shells and excess AAV2 particles. Samples were analyzed via negative stain TEM, where images were collected using an automated montage setup to minimize data collection bias. Particles were blindly quantified to estimate the number of full and empty shells for all three handle variants hybridized to heparan sulfate 3c. These experiments revealed that H1 was less effective at viral capture than the longer and denser H2 and H3 handles. For H1, only 20% of the O shells were occupied by AAV2, while for H2 and H3, capture increased to 84% and 96%, respectively ( Figure 8 b). Unless otherwise stated, the branched handle design (H3) hybridized to heparan sulfate 18-mer variant 3c will be used hereafter.

[0252] 2-Multi-virus capture and capture of different virus types in exactly the same capsid unit

[0253] To test whether our system can be used as a true broad-spectrum virus capture platform, we challenged the heparan sulfate-modified T1 half-shells to capture a viral mixture consisting of AAV2, HPV16, and Chikungunya virus particles. Negative stain TEM characterization of such samples revealed capture of all virus types present in the mixture ( Fig.21 ). Fig.21 The field micrographs in A and B illustrate the good performance of the system. Some capsids were found to encapsulate a single virus, such as a Chikungunya virus particle ( Fig.21 C) an HPV16 ( Fig.21 D) and an AAV2 ( Fig.21 E), but multiple particles were also captured simultaneously in the same manner as seen with several AAV2 ( Fig.21 F top), HPV16-Chik( Fig.21 F bottom), AAV2-Chik( Fig.21 G) and AAV2-HPV16 ( Fig.21 H) in identical shell units.

[0254] 3. Cooperative Capture of Virus Clusters

[0255] If the capsid diameter is much larger than the target virus size, we observed that multiple viral particles can be sequestered. Interestingly, multiple copies of the HS-modified capsid can separate and cover the surface of the AAV2 cluster ( Fig. 22 ).

[0256] Overview:

[0257] In summary, we present here a virus capture system that targets viral features that are conserved across many families by using HS derivatives. Overall, we achieved encapsulation of nine different virus and VLP test samples, each representing a different viral family, and varying in size and surface complexity. Our modular capsid system creates a locally curved environment within the cavity that enables highly multivalent binding and can be optimized based on size and ligand density / type to achieve a broad-spectrum antiviral platform with irreversible binding. Our capsids can bend and conform to the shape of the captured viral particle to some extent, suggesting that this capsid system can also accommodate pleomorphic viral particles.

[0258] We envision that our HS-modified DNA origami shells could act as cell surface decoys, sequestering viruses and preventing interactions with the cell surface, and thereby reducing effective viral loads in acute infections. Testing the therapeutic potential of this system to reduce viral loads in vivo remains an important future task. In addition to virus neutralization, our system could also serve as a capture agent for relevant viral proteins ( Fig. 20 ) and other byproducts, such as subviral particles that may overwhelm the immune system (Zelikin, loc. cit.; Chai, N. et al., J. Virol. 82 (2008) 7812-7817). Overall, our results strongly indicate that our heparan sulfate-modified capsid library has the potential to become a relevant therapeutic platform to combat viral infections.

Claims

1. A DNA-based nanostructure, wherein the DNA-based nanostructure is a shell comprising a cavity surrounded by the DNA-based nanostructure, wherein the DNA-based nanostructure is formed by self-assembling DNA-based building blocks, wherein each of the self-assembling DNA-based building blocks is formed by a single-stranded DNA template strand and a set of oligonucleotides complementary to the single-stranded DNA template, wherein each of the oligonucleotides is complementary to one continuous DNA sequence fragment on the single-stranded DNA template, or is complementary to at least two non-continuous DNA sequence fragments on the single-stranded DNA template, wherein each of the self-assembling DNA-based building blocks is a triangular prism and / or a rectangular prism, in particular a triangular prism, and wherein a subset of one or more of said oligonucleotides in one or more of said self-assembling DNA-based building blocks are each linked to a construct comprising at least one sulfonated or sulfated polysaccharide group, in particular a construct comprising one or two sulfonated or sulfated polysaccharide groups, directed towards the interior of said cavity, Each of the constructs comprises: (i) a handle comprising at least one binding site for the sulfonated or sulfated polysaccharide group; and (ii) the sulfonated or sulfated polysaccharide group bound to the handle; wherein the handle has a length at least corresponding to the length of a single-stranded oligonucleotide comprising 30 nucleotides.

2. The DNA-based nanostructure of claim 1, wherein each of the handles comprises two binding sites for the sulfonated or sulfated polysaccharide groups.

3. The DNA-based nanostructure according to claim 1 or 2, wherein each of the sulfonated or sulfated polysaccharide groups is independently selected from the following list: heparin, heparan sulfate, hybrid heparan sulfate, carrageenan, cellulose sulfate and dextrin 2-sulfate.

4. The DNA-based nanostructure according to claim 3, wherein each of the sulfonated or sulfated polysaccharides is independently selected from heparan sulfate and hybrid heparan sulfate, in particular heparan sulfate.

5. The DNA-based nanostructure according to any one of claims 1 to 4, wherein one or more of the self-assembling DNA-based building blocks in the subgroup comprises n single-stranded oligonucleotides as the handles, wherein each handle is independently connected to at least one of the sulfonated or sulfated polysaccharide groups, wherein n is an integer independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, in particular, wherein n is 9.

6. The DNA-based nanostructure according to any one of claims 1 to 5, wherein the handle is a single-stranded oligonucleotide having a length between 30 and 60 nucleotides, particularly between 40 and 55 nucleotides, more particularly between 45 and 50 nucleotides.

7. The DNA-based nanostructure according to any one of claims 1 to 6, wherein each of the sulfonated or sulfated polysaccharide groups comprises an oligonucleotide having a sequence complementary to an oligonucleotide fragment contained in the handle.

8. The DNA-based nanostructure according to any one of claims 1 to 7, which is a closed three-dimensional geometric shape, in particular a closed three-dimensional geometric shape selected from the following items: spheres, spherocylinders and polyhedrons, in particular tetrahedrons, octahedrons or icosahedrons, and the closed three-dimensional geometric shape is formed in situ from the self-assembling DNA-based building blocks in the presence of the virus or virus particle to be encapsulated.

9. The DNA-based nanostructure according to any one of claims 1 to 7, which is a shell having an opening for accessing the cavity.

10. The DNA-based nanostructure according to any one of claims 1 to 7, wherein the DNA-based nanostructure is a combination of a first subshell and a second subshell, wherein the first subshell and the second subshell each have an opening for respectively entering a first inner cavity and a second inner cavity, wherein the first inner cavity and the second inner cavity together form the cavity, in particular, wherein the first subshell and the second subshell are connected by at least one joint.

11. The DNA-based nanostructure according to any one of claims 1 to 10, which is based on an icosahedral structure.

12. The DNA-based nanostructure according to claim 11, wherein the DNA-based nanostructure is a DNA-based nanostructure formed by self-assembling DNA-based structural units, wherein each of the self-assembling DNA-based structural units is a triangular prism and / or a rectangular prism, in particular a triangular prism.

13. The DNA-based nanostructure according to claim 12, Each of the triangular prisms and / or rectangular prisms is formed by m triangular planes or rectangular planes, respectively, wherein m is an integer independently selected from 4, 5, 6, 7 and 8, in particular an integer independently selected from 5, 6 and 7, more particularly, wherein the integer is 6, Three or four sides of each of the m planes are respectively formed by n parallel DNA double helix fragments, wherein n is an integer independently selected from 1, 2, 3, 4, 5 and 6, particularly independently selected from 2, 3, 4 and 5, more particularly independently selected from 3 and 4, wherein each plane is connected to a plane above said plane and / or to a plane outside said plane (i) by stacking interactions between DNA double helices forming said plane and (ii) in part by DNA fragments within said single-stranded DNA template and / or said oligonucleotide forming said DNA-based building blocks bridging at least two of said planes, and At least two of the three or four side ladders contain a specific pattern of recesses and / or protrusions formed by missing or additional DNA double helix segments, respectively, for specific interaction with a complementary pattern on the side ladder of another one of the self-assembling DNA-based building blocks.

14. The DNA-based nanostructure according to any one of claims 1 to 13, wherein the DNA-based nanostructure is a half-shell selected from: (a) A semi-octahedral DNA-based nanostructure based on a T-octa self-assembling DNA-based building block, wherein the semi-octahedral DNA-based nanostructure is composed of a set of four copies of a triangular frustum, wherein inactivating base pair stacking contacts on one of the triangular sides of the triangular frustum by chain shortening or by addition of unpaired thymidine; (b) a T=1 half-shell based on a T1_pentamer_triangle self-assembling DNA-based building block, consisting of two sets of five copies in each case of two different triangular frustums, wherein the five copies of the first set form a closed pentamer and the five copies of the second set are docked to the edges of the pentamer; and (c) a "trap-like" T=1 half-shell with missing pentagonal vertices based on a combination of a T1_pentamer_triangle self-assembling DNA-based building block and a T1_ring_triangle self-assembling DNA-based building block, the "trap-like" T=1 half-shell consisting of three groups of five copies of three different triangular frustums in each case, wherein the five copies of the first group form a closed pentamer, the five copies of the second group are docked to the edge of the pentamer, the five copies of the third group are docked to the edge of the pentamer, and the five copies of the third group are docked into the gaps between the five copies of the second group; (d) T=3 icosahedral half-shell based on T3_6_triangle-like self-assembling DNA-based building blocks, which consists of a total of 30 triangular subunits, which are divided into five copies of six different full-size DNA triangle designs with specific edge docking rules.

15. The DNA-based nanostructure according to any one of claims 12 to 14, further comprising: (a) one or more types of DNA brick constructs, each type of such DNA brick constructs being characterized by one or more interaction sites for specific interaction via edge-to-edge stacking contacts with one or more complementary interaction sites present on a plane of a triangular frustum or a rectangular frustum on the outer surface of the DNA-based nanostructure, wherein the DNA brick construct covers the free space between three or four sides of the plane, respectively; (b) one or more cross-linked portions, wherein the cross-linked portions are within one of the triangular prisms or rectangular prisms and / or between two of the triangular prisms and / or rectangular prisms; and / or (c) at least one moiety that specifically interacts with the virus or viral particle.

16. A composition comprising the DNA-based nanostructure according to any one of claims 1 to 15 encapsulating one or more viruses or viral particles.

17. A method for encapsulating one or more viruses or viral particles, the method comprising the steps of: Providing a DNA-based nanostructure according to any one of claims 1 to 7 and 9 to 15, and contacting the DNA-based nanostructure with a medium containing or suspected of containing the virus or viral particle.

18. The method of claim 17, wherein (i) for a size of at most 50*50*50 nm 3 For viruses, DNA-based semi-shell nanostructures based on T_octa self-assembling DNA-based building blocks were selected; (ii) for viruses with sizes between 15*15*15 and 100*100*100nm 3 For viruses between 15*15*15 and 100*100*100 nm, a DNA-based semi-shell nanostructure based on a T1_pentamer_triangular self-assembling DNA-based structural unit was selected; (iii) for viruses with a size between 15*15*15 and 100*100*100 nm, a DNA-based semi-shell nanostructure based on a T1_pentamer_triangular self-assembling DNA-based structural unit was selected 3 For viruses between the two, a DNA-based semi-shell nanostructure based on a combination of a T1_pentamer_triangle self-assembly type DNA-based structural unit and a T1_ring_triangle self-assembly type DNA-based structural unit is selected; (iv) for a 50*50*50nm 3 For viruses larger than 1.5 μg / ml, DNA-based half-shell nanostructures based on T3_6_triangle self-assembling DNA-based building blocks were selected.

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