Broad-spectrum virus-trapping nanoshells

Abstract

The present invention relates to DNA-based nanostructures for encapsulating viruses or viral particles, compositions comprising one or more viruses or viral particles encapsulated by such DNA-based nanostructures according to the invention, and methods for encapsulating one or more viruses or viral particles by using such DNA-based nanostructures.

Description

[Technical field]

[0001] The present invention relates to DNA-based nanostructures for encapsulating a broad spectrum of viruses or viral particles, compositions comprising one or more viruses or viral particles encapsulated by such DNA-based nanostructures according to the invention, and methods for encapsulating one or more viruses or viral particles by using such DNA-based nanostructures. [Background technology]

[0002] Currently, there are more than 200 known virus-vector-mediated human diseases, of which only nine are treatable with current antiviral drugs (Heida et al., Drug Discov. Today 26 (2021) 122-137). In the search for effective antiviral therapy, neutralizing antibodies are increasingly considered to treat acute viral infections (e.g., Wang et al., Science. 2021 Aug 13; 373 (6556): eabh1766. doi: 10.1126 / science.abh1766. Epub 2021 Jul 1. PMID: 34210892; Taylor, PC et al., Nat. Rev. Immunol. 21 (2021) 382-393). Antiviral antibodies often induce virus neutralizing functions by blocking the interaction of viruses with specific receptors on the surface of host cells that are 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 virus serotype at a time. Furthermore, antibodies or other proteinaceous virus-binding agents can cause deleterious immunogenic effects in organisms, resulting in substantial additional production hurdles and costs.

[0003] Recently, a new concept of neutralizing viruses by encapsulation in polymeric shells fabricated with DNA origami has been presented (WO 2021 / 165528). The shells mechanically prevent interactions between captured viruses and host cells. For proof-of-concept experiments, the inside of the shells were coated with antibodies to sequester the virus particles within the shell. Heparin is mentioned as a potential alternative binding moiety, but no data are shown. One important advantage of the shells is that the virus-binding moieties used inside them do not themselves need to have neutralizing function, since this task is performed by the shell material. Nevertheless, as mentioned above, the use of antibodies in virus-capturing shells presents several challenges that may limit the usefulness of the virus-capturing concept. WO 2021 / 165528 used up to 90 antibodies per virus-capturing shell, attached via interactions with single-stranded oligonucleotide handles with 16-mer or 26-mer overhangs for hybridization. WO 2021 / 165528 demonstrates that viral particles can be successfully encapsulated in shells with antibodies, but does not quantify the encapsulation rate achieved.

[0004] The concept described in WO 2021 / 165528 has also been described in a scientific publication (Sigl et al., Nat. Mater. 20 (2021) 1281-1289). Sigl et al. show successful encapsulation of virus particles using virus particles equipped with antibodies. Heparin is not mentioned as an alternative binding moiety, and no quantification of the encapsulation rate is provided.

[0005] Knappe et al. (ACS Nano 2021;14316-14322) describe DNA origami particles functionalized via click chemistry, so that different types of functional moieties, including antibodies and carbohydrates, can be attached to the DNA origami particles. Heparin is not specifically mentioned in Knappe et al., and since the DNA origami particles are closed shells, the functionalization is performed on the outside of the DNA origami particles. Therefore, no information can be derived from Knappe et al. regarding the options for encapsulating viral particles inside the DNA origami shell, as well as any peculiarities of the optimal design of the attachment sites.

[0006] Another attractive avenue is the packaging of viral payloads for the purpose of delivering viruses or other vectors for genetic information or other cargo to target cells or tissues, as discussed above, for example in Antigen-Triggered Logic-Gating of DNA Nanodevices, Engelen et al., J. Am. Chem. Soc. 2021, 143, 51, 21630-21636, December 20, 2021. Also, here, the use of antibodies as moieties for attaching viruses or virus-like particles or other aggregates in a DNA-based shell may limit the scope of such applications due to the above-mentioned challenges arising from the use of antibodies.

[0007] Thus, although various strategies for the treatment of viral infections have been developed or proposed, there is still a need to develop a concept of a universal antiviral drug platform to target various viral pathogens. In particular, a concept that does not depend on prior detailed knowledge of the genetics and properties of the target virus would be highly desirable. Furthermore, there is an unmet need for the development of a system that allows the encapsulation of viral particles with high efficiency. Summary of the Invention

[0008] It is an object of the present invention to provide constructs that allow for the encapsulation of one or more viruses or viral particles, the solution to which is to say the use of polymeric components such as DNA-based nanostructures has not yet been taught or suggested by the prior art.

[0009] Thus, in one aspect, the present invention relates to a DNA-based nanostructure, which is a shell with a cavity surrounded by the DNA-based nanostructure, which is formed by self-assembling DNA-based building blocks, each of which is formed by a single-stranded DNA template strand and a set of oligonucleotides complementary to the single-stranded DNA template, each of which is complementary to either one contiguous DNA sequence stretch or at least two discontinuous DNA sequence stretches on the single-stranded DNA template, each of which is A triangular prism and / or a rectangular prism, particularly a triangular prism, wherein one or more subsets of said oligonucleotides within 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 pointing into the interior of said cavity, particularly a construct comprising one or two sulfonated or sulfated polysaccharide groups, each said construct comprising (i) a handle comprising at least one binding site for said sulfonated or sulfated polysaccharide group, and (ii) said sulfonated or sulfated polysaccharide group(s) attached to said handle, said handle having a length corresponding to the length of a single-stranded oligonucleotide comprising at least 30 nucleotides.

[0010] In a second aspect, the present invention relates to a composition comprising a DNA-based nanostructure according to the 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 said DNA-based nanostructure with a medium containing or suspected of containing said viruses or viral particles.

[0012] The present disclosure contemplates 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 described in the detailed description and examples.

[0013] Other features, objects, and advantages of the compositions and methods herein will become apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0014] [Figure 1]DNA origami shells and their functionalization with HS derivatives. A: Heparan sulfate proteoglycans (HSPGs) interact with viral pathogens and mediate their cellular uptake (left). Schematic of a DNA origami shell with an internal HS modification that can bind and capture viral particles (right). B: SPAAC reaction between azide-modified HS oligomers and DBCO-modified DNA oligos. The DNA sequences are complementary to the handles of the DNA origami shells in (e, f). C: PAGE characterization of HS-modified DNA oligos. Products containing HS with sulfate and sulfonate groups (3a and 3c) migrate through the gel at a faster rate than the analogue negative controls (3b and 3d) due to their increased anionic nature. D: Cylindrical models of O and T1 shells, consisting of 4 and 10 triangular subunits, respectively, containing single-stranded protruding oligos (shown in red, called handles) decorating their interiors. Each triangular subunit contains 9 handle positions. E: A new T3 shell design consisting of 30 triangular subunits and featuring an internal cavity of 150 nm. Each triangular subunit also contains 9 handle locations. F: Negative stain TEM micrograph of a T3 shell. Scale bar is 100 nm. G: Schematic of three different handle designs. H1 contains one HS modification per handle positioned as close as possible to the origami surface. H2 also contains one HS modification per handle but has a 20 base poly-T stretch, allowing the handles to reach further than H1. H3 mimics a branched polymer containing two HS modifications per handle unit, thus doubling the local HS density. [Diagram 2] Showing viruses and VLPs entrapped within HS modified O, T1 and T3 shells. Negative stain TEM captions of: a: AAV2, Polio3, mature Dengue1 and Norovirus GII.4 successfully entrapped in O shell; b: HPV16, SARS-CoV-2, Chikungunya and Rubella encapsulated by T1 shell; c: Adenovirus 5 entrapped by T3 shell. Scale bar is 100 nm. [Diagram 3]Showing multiple viruses and VLPs entrapped in HS-modified O, T1 and T3 shells. Negative stain TEM images of: a: Up to 4 AAV2 in one O shell; b: Up to 3 HPV16 in one T1 shell; c: One HPV16 coordinated by two O shells for complete occlusion of the viral particle; d: Up to 6 AAV2 per T1 shell; e: Up to 3 Chikungunya VLPs per T3 shell; f: Cooperative effect of multiple O shells to entrap large numbers of AAV2 particles. Scale bar is 100 nm. [Figure 4] Cryo-EM analysis of virus-like particles trapped in DNA origami shells. a: Cryo-EM micrograph of O-shell binding to HPV16 VLP. b: 2D class average images of one or two O-shells binding to one HPV16 particle, showing different orientations of the complex. White arrows indicate the gap difference between the two O-shells, confirming the capture of VLP particles of different sizes. c: 3D reconstruction of HPV16 bound to one and two O-shells. d: Cryo-EM micrograph of T1 shell binding to Chikungunya VLP. e: 2D class average images of T1 shell bound to Chikungunya particle showing different orientations of the complex. f: Two different views of 3D reconstruction of T1 shell encapsulating Chikungunya virus particle. [Diagram 5] SPAAC reaction of 8-mer HS derivatives (1a and 1b). a: Click chemistry reaction between azide-modified HS polymer and DBCO-modified DNA oligo. The DNA sequence is complementary to the handle of the DNA origami shell. b: PAGE characterization of all HS-modified DNA oligos. [Figure 6] The T3 shell design is shown in a: Top and front views of the T3 cylindrical model, b: Cylindrical model of triangles t1-t6 included in the T3 shell assembly, arrows indicate complementary lateral interactions. [Figure 7] TEM view of T3 shells. The T3 DNA origami shells exhibited an inner diameter of approximately 150 nm. Due to their flexibility, they appear to deform on the grid. Scale bar is 400 nm. [Figure 8]Figure 1 shows TEM quantification of O-shells for AAV2 capture with different handle designs. a: Schematic of three different handle designs H1, H2 and H3. HS is represented as a red hexagon. H1 contains one HS modification per handle, located close to the origami surface. H2 also contains one HS modification per handle, but has a 20 base poly-T stretch, allowing the handles 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-to-air O-shells for each handle design when functionalized with 3c HS derivatives and AAV2 in excess. H1 showed ∼20%, H2 ∼84% and H3 ∼96% full shells. c: Schematic of O half shells and their ssDNA handles within the internal cavity. [Figure 9] 3d TEM of a negative control for AAV2 capture in the O shell. 3d Two fields of the same sample showing that no binding was observed when the negative control HS modification was hybridized to the H3 handle design. Scale bar is 100 nm. [Figure 10] Figure 1 shows a TEM of AAV2 capture by origami shell excess. Two fields of the same sample showing that all AAV2 particles were encapsulated when an excess of origami shells was used. Scale bar is 100 nm. [Figure 11] TEM of free virus and VLPs are shown. TEM data showed that AAV2, Poliovirus, HPV16, Chikungunya and Adenovirus 5 were the purest samples of our library. Dengue, Norovirus, SARS-CoV-2 and Rubella contained large amounts of visible protein debris and showed a variable range of particle sizes. Scale bar is 100 nm. [Figure 12] TEM tomography of adenovirus 5 within a T3 shell. Tomogram slices calculated from the EM tilt series demonstrated full encapsulation of adenovirus into selected shell particles. Scale bar is 100 nm. [Figure 13]TEM tomography of chikungunya VLPs in a T3 shell. Tomogram slices calculated from the EM tilt series demonstrated the full encapsulation of three chikungunya VLPs in the selected shell particle. The last image slice showed a disruption of the triangular connectivity. It was not clear whether this discontinuity was due to a repositioning of the shell to encapsulate multiple VLPs or a consequent deformation on the grid during sample preparation. Scale bar is 100 nm. [Figure 14] TEM quantification of T1 shells capturing Chikungunya VLPs with 3a and 3b HS derivatives. a: Negative stain TEM micrograph of T1 shells functionalized with 3b negative control HS derivative. Due to size and shape complementarity, the weak electrostatic interaction between DNA and Chikungunya VLPs was sufficient to keep the virus particles encapsulated when the negative control handle was used. Scale bar is 100 nm. b: TEM quantification of full vs empty T1 shells functionalized with 3a HS derivative in H1 handle design. Approximately 90% of the shells were full. c: TEM quantification of full vs empty T1 shells functionalized with 3b negative control HS derivative in H1 handle design. Approximately 54% of the shells were full. [Figure 15] TEM of immature and mature dengue 1 VLPs captured in O-shells. a: The immature configuration of dengue VLPs showed no binding to the HS-modified origami shell. b: Mature dengue VLPs were recognized and encapsulated by the O-shell. VLPs were used in excess. Scale bar is 100 nm. [Figure 16]Cryo-EM imaging of O-shells trapping HPV16 particles (EMD-13884). a: Exemplary micrograph of O-shells trapping HPV16 vitrified on a lacy carbon grid with ultrathin carbon support. b: 2D class average of empty shell (left), HPV16 trapped by one O-shell (middle), and two O-shells trapping HPV16 (right). c: 3D class of selected particles showing similar particle as in b. d: 3D reconstruction of HPV16 particle trapped in one O-shell. e: Multibody refinement of HPV16 particle encapsulated by two O-shells. f+g: Multicomponent analysis of two O-shells trapping HPV16. [Figure 17] Cryo-EM imaging of T1 shells capturing Chikungunya VLPs (EMD-13883). a: Exemplary micrograph of T1 shells capturing Chikungunya VLPs vitrified on a lacy carbon grid with an ultrathin carbon support. b: 2D class average of extracted particles. c: FSC estimate of the reconstruction shown in e (C5). d: 3D classification of extracted particles. e: 3D reconstruction of Chikungunya-capturing T1 shells of particles selected from multiple 3D classifications without (C1) and with (C5) symmetry. [Figure 18] Figure 2 shows 2D class averages of free HPV16 VLPs extracted from cryo-EM images. The diameter of HPV16 VLPs ranged from 35 nm to 50 nm. [Figure 19] Figure 1 shows the stability of virus capture by the shell. TEM quantification of AAV2 capture in O shells subjected to a dilution series. The percentage of occupied shells remained the same before and after 100-fold dilution and 14 days of incubation at room temperature in diluted samples compared to undiluted samples. The overall shell concentration was 0.072 nM. [Figure 20] TEM of rubella protein debris trapped in T1 shell. Successful encapsulation of protein debris from a rubella VLP sample. [Figure 21]Negative staining TEM imaging results of viral cocktail capture using heparan sulfate modified T1 half shells. (A, B) TEM fields of T1 half shells capturing AAV2, Chikungunya and HPV16 viral particles in different ratios with homogeneous and heterogeneous complexes. Captured viral particles selected as (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. [Figure 22] The cooperative effect of multiple O-shells to capture multiple AAV2 particles is shown. Scale bar: 50 nm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present disclosure provides constructs that allow for encapsulation of a virus or viral particle.

[0016] Unless otherwise defined, 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.

[0017] The terms "comprising" and "including" are used herein in their open-ended and non-limiting sense, except where expressly stated. Thus, with respect to such latter embodiments, the term "comprising" includes the narrower term "consisting of."

[0018] In the context of describing the present invention (particularly in the context of the following claims), the terms "a," "an," and "the" and similar referents should be construed to encompass both the singular and the plural, unless otherwise indicated in the specification or clearly contradicted by context. For example, the term "cell" includes a plurality of cells (including mixtures thereof). When the plural is used for compounds, salts, etc., this is construed to mean a single compound, salt, etc.

[0019] Thus, in one aspect, the present invention relates to a DNA-based nanostructure, which is a shell with a cavity surrounded by the DNA-based nanostructure, the DNA-based nanostructure being formed by self-assembling DNA-based building blocks, each of which is formed by a single-stranded DNA template strand and a set of oligonucleotides complementary to the single-stranded DNA template, each of which is complementary to either one contiguous DNA sequence stretch or at least two discontinuous DNA sequence stretches 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 one or more subsets of the oligonucleotides in one or more of the self-assembling DNA-based building blocks are each linked to a construct comprising at least one sulfonated or sulfated polysaccharide group facing the interior of the cavity, in particular a construct comprising one or two sulfonated or sulfated polysaccharide groups, each of the constructs comprising (i) a handle comprising at least one binding site for the sulfonated or sulfated polysaccharide group, and (ii) the sulfonated or sulfated polysaccharide group(s) attached to the handle.

[0020] In an alternative embodiment, a subset of one or more of the oligonucleotides in one or more of the self-assembling DNA-based building blocks are linked to a construct comprising at least one sialic acid group pointing towards the interior of the cavity, in particular a construct comprising one or two sialic acid groups.

[0021] In certain embodiments, the handle has a length at least equivalent to the length of a single stranded oligonucleotide comprising 30 nucleotides.

[0022] By using such extended versions of the linking moieties, we were able to surprisingly show a dramatic increase in the number of DNA-based nanostructures that actually encapsulated viral particles.

[0023] In the context of this disclosure, the term "DNA-based nanostructure" refers to a nanostructure formed by a set of DNA-based polymers. DNA-based nanostructures of the type used in accordance with the present invention are described in detail in WO 2021 / 165528 and Sigl et al., loc.cit.

[0024] In the context of this disclosure, the term "DNA" refers to a deoxyribonucleic acid composed of a single strand of monomeric units called nucleotides, each nucleotide consisting of a nitrogenous nucleobase, a 2-deoxyribose sugar moiety, and a phosphate group, with the individual nucleotides linked in a single strand by a phosphate group linking the 5' OH group of a 2-deoxyribose sugar moiety to the 3' OH group of an adjacent 2-deoxyribose sugar moiety. In certain embodiments, the nitrogenous nucleobases are independently selected from cytosine [C], guanine [G], adenine [A], and thymine [T]. In certain embodiments, the one or more nucleobases are non-standard bases, in particular modified adenosine, in particular N6-carbamoyl-methyladenine or N6-methyladenine; modified guanine, in particular 7-deazaguanine or 7-methylguanine; modified cytosine, N4-methylcytosine, 5-carboxylcytosine, 5-formylcytosine, 5-glycosylhydroxymethylcytosine, 5-hydroxycytosine, or 5-methylcytosine; modified thymidine, in particular α-glutamylthymidine or α-putresinylthymine; uracil or a variant thereof, in particular uracil, base J, 5-dihydroxypentauracil; or 5-hydroxymethyldeoxyuracil; deoxyarchaeosine and 2,6-diaminopurine. A single-stranded stretch of DNA can interact with a complementary stretch of DNA through complementary nucleobase interactions, with cytosine and guanine, and adenine and thymine being complementary to each other by forming two (A / T) and three (G / C) hydrogen bonds between the nucleobases, respectively. The two single strands of DNA can be fully complementary to each other, as in the case of genomic DNA, or they can be partially complementary to each other, including situations where one single strand of 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 duplex.

[0025] As is well known, DNA has evolved in nature as a carrier of genetic information that codes for proteins. DNA further includes non-coding regions, including regions with regulatory functions. Thus, any DNA-based application usually depends crucially on a specific DNA sequence and is in most cases only possible 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 be present, since the underlying DNA sequence is solely designed and selected such that the desired arrangement of double helical subunits is formed. Thus, in one embodiment, any form of long single-stranded DNA sequence, whether naturally occurring DNA (such as bacteriophage DNA) or synthetically produced DNA, may be selected as a template to design a set of short single-stranded DNA sequences, each sequence being complementary to one or more different portions of the template, thus forming one or more double helical portions. Taken together, all such double helical portions created by the interaction of the complete set of short single-stranded DNA sequences with the template form the desired three-dimensional arrangement. Starting from a given single-stranded template sequence, the design of a set of complements can be set up using known techniques, such as methods described for the synthesis of megadalton-scale discrete objects with structurally defined 3D shapes (18, 24-35). In particular, iterative design using caDNAno (37) coupled with elastic network-guided molecular dynamics simulations (38) can be used.

[0026] In addition to the interaction of complementary nucleobases of different stretches of single-stranded DNA via hydrogen bonds, further interactions between different DNA strands are possible, including stacking interactions between blunt ends of double-stranded DNA helices (36), thus allowing the design and formation of complex DNA-based nanostructures via shape complementarity of double-stranded helical subunits. Thus, two three-dimensional configurations formed according to the previous paragraph may interact with each other by stacking interactions between double-helical subunits present on the two three-dimensional configurations, including specific interactions between two three-dimensional configurations with complementary protrusions and recesses (or knobs and holes), as shown, for example, in Figures 7-13D of WO 2021 / 165528.

[0027] In another aspect, the present invention relates to polymer-based nanostructures which are RNA-based nanostructures.

[0028] In the context of this disclosure, the term "RNA" refers to a ribonucleic acid composed of a single strand of monomeric units called nucleotides, each nucleotide being composed of a nitrogenous nucleobase, a ribose sugar moiety, and a phosphate group, with the individual nucleotides linked in a single strand by a phosphate group linking the 5' OH group of the ribose sugar moiety to the 3' OH group of the adjacent ribose sugar moiety. In certain embodiments, the nitrogenous nucleobases are independently selected from cytosine [C], guanine [G], adenine [A], and uracil [U]. In certain embodiments, one or more nucleobases are non-standard bases, in particular non-standard bases selected from the list of pseudouridine, ribothymidine, and inosine. Unlike DNA, RNA is mostly in single-stranded form, but the formation of a double-stranded form is possible through the interaction of complementary nucleobases, with cytosine and guanine, and adenine and uracil being complementary to each other by forming two (A / U) and three (G / C) hydrogen bonds between the nucleobases, respectively.

[0029] In the context of the present invention, the term "cavity" refers to the space enclosed by the DNA-based nanostructure. In certain embodiments, the cavity resembles a sphere with a spherical segment cut off, the cut surface being formed by the self-assembling DNA-based building blocks at the boundary of the DNA-based nanostructure. In certain embodiments, the cut surface is a large circle, such that the DNA-based nanostructure is a half-shell. In other cases, when the three-dimensional geometric shape of the DNA-based nanostructure originates from a sphere-cylinder or polyhedron, in particular a tetrahedron, an octahedron or an icosahedron, the cavity should be understood as the space resulting from cutting the corresponding sphere-cylinder or polyhedron by a plane, the cut surface being formed by the self-assembling DNA-based building blocks at the boundary of the DNA-based nanostructure.

[0030] In certain other embodiments, the DNA-based nanostructure resembles a spherical segment, in which case only the portion of the virus that interacts with the DNA-based nanostructure is covered, and thus encapsulation of one or more viruses or viral particles according to the present invention requires the attachment of two or more of such DNA-based nanostructures to the one or more viruses or viral particles.

[0031] In the context of the present application, the term "sulfonated or sulfated polysaccharide group" relates to a group comprising a polysaccharide which comprises at least one sulfated hydroxy group or at least one sulfonated glycosylamino group.

[0032] Importantly, in addition to targeting specific receptors, many viruses also weakly interact with different biological substances, including sulfonated polysaccharides (Cagno, V. et al., Viruses 11 (2019) 596; see Table 2).

[0033] In certain embodiments, each of the handles comprises two binding sites for the sulfonated or sulfated polysaccharide groups.

[0034] In certain embodiments, each of the polysaccharides comprising at least one sulfated hydroxy group or at least one sulfonated glycosylamino group is independently selected from the list of heparin, heparan sulfate, hybrid heparan sulfate, carrageenan, cellulose sulfate, and dextrin 2-sulfate, in particular heparan sulfate or hybrid heparan sulfate.

[0035] In certain embodiments, the polysaccharide comprising at least one sulfated hydroxy group or at least one sulfonated glycosylamino group consists of 3 to 10 disaccharide units, in particular 4, 5, 6, 7, 8 or 9 units, in particular 4 or 9 monosaccharide units.

[0036] In certain embodiments, the disaccharide unit comprises 2 or 3 O- and / or N-sulfonate groups, in particular 3 O- and / or N-sulfonate groups per disaccharide unit.

[0037] In certain embodiments, each of the sulfonated or sulfated polysaccharide groups is independently selected from the list of heparin, heparan sulfate, hybrid heparan sulfate, carrageenan, cellulose sulfate, and dextrin 2-sulfate.

[0038] In certain such embodiments, each of the sulfonated or sulfated polysaccharides is independently selected from heparan sulfate and hybrid heparan sulfate, in particular heparan sulfate.

[0039] 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 are composed of repeating 1→4 linked disaccharide units, one monosaccharide being an α-D-glucosamine residue and the other being uronic acid (or, in salt form, uronate). Heparin is a structurally similar polysaccharide found in mast cells as a component of serglycin proteoglycan. Heparan sulfate and heparin can be defined as follows: first, in heparin, the uronate is mainly α-L-iduronate, whereas in heparan sulfate, the uronate is mainly β-D-glucuronate, the C-5 epimer of α-L-iduronate. Second, in heparan sulfate, the D-glucosamine residues are predominantly N-acetylated, whereas in heparin they are N-sulfonated. Finally, at least 70-80% of heparin is composed of the disaccharide L-iduronic acid 2-O-sulfate α(1→4) D-glucosamine N,6-sulfate in heparan sulfate, whereas approximately 40-60% of the disaccharide is composed of (1→4) D-glucuronic acid β(1→4) D-glucosamine, which may be either N-acetylated or N-sulfonated. Taken together, these structural features make heparin more sulfated and therefore more charged than heparan sulfate. However, it has become clear that the designation heparin or heparan sulfate is not as clear-cut as this description suggests, and 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 such hybrids having a structure that is a mixture of "classic" heparin structural elements (L-iduronate, highly sulfonated) and "classic" heparan sulfate structural elements (D-glucuronic acid, N-acetylated and 6-O-sulfonated).

[0040] 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 appear to be generally beneficial for the viral life cycle. For example, HSPG-virus interactions may enable infection-enhancing diffusive search of virus particles for their specific host cell receptors on the surface of cells (Figure 1A, left panel). The interaction of heparan sulfate (HS) with viruses has already been exploited for medical purposes, for example in virus-trapping coatings of 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 create 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.). In general, a high level of multivalency is required to increase the binding strength of HS-nanoparticles with viruses. The reversible nature of the binding may result in the undesired release of unbound and infectious virus from the virus-capturing coating, or the need to maintain high concentrations of therapeutically active agents (Zelikin, loc.cit.).

[0041] In certain embodiments, the subset of self-assembling DNA-based building blocks consists of 1-100% of all self-assembling DNA-based building blocks that form the DNA-based nanostructure. In certain embodiments, the subset of self-assembling DNA-based building blocks consists of 50-100%, more particularly 75-100%, and especially 100% of all self-assembling DNA-based building blocks that form the DNA-based nanostructure.

[0042] In certain embodiments, one or more of the self-assembling DNA-based building blocks in a subset comprises n single-stranded oligonucleotides as the handles, each handle independently linked to at least one of the sulfonated or sulfated polysaccharide groups, where n is an integer independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and in particular n is 9.

[0043] In a particular embodiment, the handle is a single-stranded oligonucleotide having a length of 30 to 60 nucleotides, in particular 40 to 55 nucleotides, more particularly 45 to 50 nucleotides.

[0044] Examples of self-assembling DNA building blocks in the form of truncated cones, where each small base of the truncated cone comprises 9 of said polynucleotides, such as T_octa self-assembling DNA-based building blocks, T1_pentamer_triangle self-assembling DNA-based building blocks, T1_ring_triangle self-assembling DNA-based building blocks or T3_6_triangle-based self-assembling DNA-based building blocks, are given in the examples.

[0045] In certain embodiments, each member of the n oligonucleotides comprises at least one oligonucleotide extension as a binding site, and each of the sulfonated or sulfated polysaccharide groups comprises an oligonucleotide having a sequence complementary to one of the oligonucleotide stretches contained in the handle. In certain embodiments, each member of the n oligonucleotides comprises one or two oligonucleotide stretches as a binding site.

[0046] In certain embodiments, each member of the n oligonucleotides comprises two oligonucleotide stretches as binding sites.

[0047] By using oligonucleotides containing two binding sites, it was surprisingly possible to show that the number of DNA-based nanostructures that actually encapsulated viral particles was further increased, even when compared to the already favorable version with the extended handle design described above. This is particularly surprising in light of the fact that the additional second binding site is in the same position as the initial binding site of the H1 handle design with a short oligonucleotide (26-mer) and that the addition of the second binding site and the HS moiety could have been expected to reduce the encapsulation efficiency due to steric hindrance.

[0048] Examples of nine subsets of such oligonucleotide handles that can be linked to constructs comprising sulfonated or sulfated polysaccharide groups can be found in the set of Hx oligos 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 oligo comprising one handle, and in the set of Hx oligos according to SEQ ID NOs: 195-203, 407-415 and 625-633, each oligo comprising two binding sites.

[0049] In certain embodiments, the cavities have 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.

[0050] In certain embodiments, the cavities have a diameter of up to 1,000 nm.

[0051] In the context of the present invention, the term "diameter" refers to the diameter of the smallest circle encompassed by the surface of a DNA-based nanostructure. For clarity, in the case of a DNA-based nanostructure in the form of a capsule (or a spherocylinder), the diameter is the diameter of the hemispherical end and / or the diameter of the cylindrical center.

[0052] In certain embodiments, the DNA-based nanostructure has a molecular mass 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 mass of at least 50 MDa, at least 80 MDa, at least 100 MDa, at least 200 MDa, or at least 500 MDa. In certain embodiments, the DNA-based nanostructure has a molecular mass of up to 1,500 MDa.

[0053] In certain embodiments, the molecular mass (MDa) of a DNA-based nanostructure and the volume (nm) of a cavity enclosed by the DNA-based nanostructure are 3 ) is less than 10,000, particularly less than 9,000. In a particular embodiment, the ratio has a value of 1,000 to 10,000, particularly 2,000 to 9,000. For example, when the molecular weight is about 40 MDa and the coating volume is about 113,000 nm 3 For one particular octahedral nanostructure, where θ is the ratio is about 2,800.

[0054] In particular embodiments, the ratio of the external surface area of ​​the DNA-based nanostructure covered by the macromolecules forming said DNA-based nanostructure to the external surface area not covered by said macromolecules (excluding the area of ​​the openings of the DNA-based nanostructure in the form of a shell) is at least 1, in particular at least 2, in particular at least 4, in particular at least 6, in particular at least 8. In other particular embodiments, the ratio is at least 10. In particular embodiments, the ratio is between 1 and 20, in particular between 2 and 18, between 4 and 16, between 6 and 14, more particularly between 8 and 12. For example, if the DNA-based nanostructure is a hemispherical shell, only the area of ​​the curved surface, i.e. the area of ​​the flat surface of the hemisphere, and not the openings, is used to calculate said ratio.

[0055] In certain embodiments, the molecular weight of each self-assembling DNA-based building block is between 4.5 and 5.5 MDa.

[0056] In certain embodiments, each self-assembling DNA-based building block comprises between 7,500 and 8,500 base pairs.

[0057] In certain embodiments, the DNA-based nanostructure consists of between 4 and 180 such self-assembling DNA-based building blocks.

[0058] In certain embodiments, the single-stranded DNA template is or is derived from the single-stranded DNA of a filamentous bacteriophage.

[0059] In the context of the present invention, the term "filamentous bacteriophage" refers to a type of bacteriophage or bacterial virus characterized by its filamentous shape that usually contains a genome of circular single-stranded DNA and infects Gram-negative bacteria. Filamentous phages include M13, Ff phages such as f1 and fd1 phages, and Pf1 phage.

[0060] 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.

[0061] In the context of the present invention, a single-stranded DNA template "derived from the single-stranded DNA of a filamentous bacteriophage" refers to a DNA construct derived from the naturally occurring published DNA sequence of a filamentous bacteriophage by one or more of the following: (i) opening a circular structure into a linear sequence, (ii) deletion of one or more nucleotides, (iii) insertion of one or more nucleotides, (iii) substitution of one or more nucleotides, (iv) addition of one or more nucleotides, and (v) modification of one or more nucleotides. Any such changes may have deleterious or at least rather unpredictable effects on the bacteriophage biology, its infectivity and its propagation ability, but 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 the need to have any functional properties, and all structural aspects such as the correct formation of the three-dimensional shape of the self-assembling DNA-based building block are implemented by the appropriate selection of the set of complementary oligonucleotides.

[0062] In certain embodiments, the single-stranded DNA template has at least 80%, in particular at least 90%, more particularly at least 95% sequence identity to a naturally occurring or published sequence of a filamentous bacteriophage, in particular a sequence selected from the sequences of M13, f1 or fd1 phages, in particular SEQ ID NO: 1 (M13 8064) and M13 7249 (SEQ ID NO: 2 of WO 2021 / 165528).

[0063] In certain embodiments, DNA-based nanostructures are closed three-dimensional geometric shapes, particularly selected from spheres, spherocylinders, and polyhedra, particularly tetrahedrons, octahedrons, or icosahedrons, that are formed in situ from the self-assembling DNA-based building blocks in the presence of the encapsulated virus or viral particle.

[0064] In certain 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-stranded DNA template.

[0065] In certain other embodiments, the self-assembling DNA-based building blocks that form the DNA-based nanostructure in situ are a collection of one or more pre-assembled DNA-based building blocks consisting of two or more individual DNA-based building blocks, each of which contains a single-stranded DNA template.

[0066] In certain embodiments, all of the self-assembling DNA-based building blocks are preassembled DNA-based building blocks. In alternative embodiments, the self-assembling DNA-based building blocks are a mixture of preassembled DNA-based building blocks and individual DNA-based building blocks, each of which includes one single-stranded DNA template.

[0067] In such embodiments, the preassembled DNA-based building blocks form a curved geometric shape and the handle or the construct comprising at least one sulfonated or sulfated polysaccharide group linked to the handle resides in the negative curvature of the curved geometric shape, such that the handle or construct is oriented toward the interior of a cavity formed from the self-assembly of the preassembled DNA-based building blocks.

[0068] In another particular embodiment, the DNA-based nanostructure is a shell having an opening to access the cavity.

[0069] 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;

[0070] In yet another particular embodiment, the DNA-based nanostructure is a combination of a first subshell and a second subshell having openings to access first and second internal cavities, respectively, which together form the cavity.

[0071] In certain embodiments, the first and second subshells are connected by at least one linker.

[0072] In certain embodiments, the linker is a linker selected from a DNA linker, an RNA linker, a polypeptide linker, a protein linker, and a chemical linker.

[0073] In the context of the present invention, the term "DNA linker" refers to a linker formed from DNA, the sequence of which is not complementary to either the DNA of the single-stranded DNA template or to the set of oligonucleotides complementary to the single-stranded DNA template, and which is linked at one end to a DNA sequence that forms the self-assembling DNA-based component of the first shell and at the other end to a DNA sequence that forms the self-assembling DNA-based component of the second shell.

[0074] In the context of the present invention, the term "polypeptide linker" refers to a linker formed from at least two, in particular at least 5, at least 10 or at least 20 amino acid residues linked by peptide bonds, the polypeptide having no tertiary or quaternary structure, the polypeptide linker being linked at one end to a DNA sequence that forms the self-assembling DNA-based component of the first shell and at the other end to a DNA sequence that forms the self-assembling DNA-based component of the second shell.

[0075] In the context of the present invention, the term "protein linker" refers to a linker formed of at least 20, in particular at least 50, at least 100, at least 200, at least 500 or at least 1,000 amino acid residues, in particular less than 1,500 amino acid residues, linked by peptide bonds, said polypeptide having a tertiary and / or quaternary structure, said protein linker being linked at one end to a DNA sequence forming the self-assembling DNA-based component of said first shell and at the other end to a DNA sequence forming the self-assembling DNA-based component of said second shell. In certain embodiments, said protein linker is covalently attached to said DNA sequence. In certain other embodiments, said protein linker is non-covalently attached to said DNA sequence, in particular said protein linker is an antibody-based protein linker, in particular selected from diabodies and full antibodies, including IgG antibodies.

[0076] In the context of the present invention, the term "chemical linker" refers to a continuous chain of 1 to 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) in its backbone; thus, in the context of the present invention, the term "between" is used to include the referenced boundary), i.e., the length of the linker is defined as the shortest connection 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, the chemical linker is preferably a C 1~20 -Alkylene group, C 1~20 -heteroalkylene group, C 2~20 -alkenylene group, C 2~20 -heteroalkenylene group, C 2~20 -alkynylene group, C 2~20 -heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, or heteroaralkylene, which may be optionally substituted. The linker may contain one or more structural elements, such as carboxamide, ester, ether, thioether, disulfide, urea, thiourea, or hydrocarbon moiety. The linker may contain two or more combinations of these structural elements. Each of these structural elements may be present more than once in the linker, for example, two, three, four, five, or six times. In some embodiments, the linker may contain a disulfide bond. It is understood that the linker must be attached to the two DNA sequences linked by the chemical linker, either in a single step or in two or more subsequent steps. For that purpose, the linker used will preferably have two groups at the proximal and distal ends which are either (i) capable of forming a covalent bond with a group present on one of the two DNA sequences to be linked, or (ii) activated to form a covalent bond with one of the two DNA sequences, or which can be activated.

[0077] In certain embodiments, the DNA-based nanostructures are based on icosahedral structures.

[0078] In certain embodiments, each of the self-assembling DNA-based building blocks is a prism.

[0079] In the context of the present invention, the term "prismoid" refers to a polyhedron with all vertices lying in two parallel planes.

[0080] In certain embodiments, the prism is a triangular prism, while in other embodiments, the prism is a rectangular parallelepiped.

[0081] In certain embodiments, the DNA-based nanostructures are based on a mixture of triangular prisms and rectangular prisms.

[0082] In certain embodiments, the present invention relates to DNA-based nanostructures, each said triangular prism or said rectangular parallelepiped is formed by m triangular or rectangular planes, respectively, where m is an integer independently selected from 4, 5, 6, 7 and 8, in particular independently selected from 5, 6 and 7, more particularly the integer is 6; three or four edges of each of the m planes are respectively formed by n parallel stretches of DNA double helices, where 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; each plane is connected to a plane above and / or beyond the plane in part by (i) stacking interactions between the DNA double helices forming the plane, and (ii) by DNA stretches within the single-stranded DNA template and / or the oligonucleotides forming the DNA-based building blocks that bridge at least two of the planes; At least two of the three or four lateral trapezoids each contain a specific pattern of recesses and / or protrusions formed by missing or additional DNA double helix stretches due to specific interactions with complementary patterns on another lateral trapezoid of the self-assembling DNA-based component.

[0083] In a particular embodiment, the average length of each of the n stretches of DNA double helices in the m planes of each of the triangular prism or rectangular prism is between 80 and 200 base pairs.

[0084] In certain embodiments, the triangular prism is a truncated pyramid, and in certain embodiments, the rectangular parallelepiped is a truncated square pyramid.

[0085] In the context of the present invention, the term "frustum of a triangular pyramid" refers to a three-dimensional geometric shape in the form of a triangular pyramid, and the term "frustum of a square pyramid" refers to a three-dimensional geometric shape in the form of a square pyramid, the tip of which has been removed to leave an upper plane parallel to the base of the pyramid.

[0086] In certain embodiments, for at least a portion of the self-assembling DNA-based building blocks, at least one edge of each of the m planes decreases in length from a first plane to the mth plane, resulting in a Behel angle between a plane perpendicular to the first plane and the trapezoid plane formed by the m edges (see FIG. 5 of WO 2021 / 165528). In certain embodiments, three or all four trapezoid planes, respectively, exhibit a Behel angle.

[0087] In a particular embodiment, the Beher angle is between 16° and 26°, particularly between 18° and 24°, more particularly between 20° and 22°, and most particularly about 20.9°.

[0088] In certain embodiments, the DNA-based nanostructure comprises at least one set of self-assembling DNA-based building blocks, where every three or four sided trapezoid comprises a specific pattern of recesses and / or protrusions formed by missing or additional DNA double helix stretches due to specific interactions with complementary patterns on another one of the sided trapezoids of the self-assembling DNA-based building blocks, respectively.

[0089] In certain embodiments, particularly in the case of closed three-dimensional geometries of DNA-based nanostructures, the self-assembling DNA-based building blocks are all identical.

[0090] In certain embodiments, the DNA-based nanostructure comprises two or more sets of self-assembling DNA-based building blocks.

[0091] In certain embodiments, the DNA-based nanostructures are rod-shaped.

[0092] In certain embodiments, the DNA-based nanostructure comprises two or more sets of self-assembling DNA-based building blocks.

[0093] In certain such embodiments, the rod-shaped DNA-based nanostructure comprises at least a first and a second set of self-assembling DNA-based building blocks, the first and second sets differing at least with respect to the Vehel angle. In certain embodiments, at least one set consists of self-assembling DNA-based building blocks exhibiting only two Vehel angles. In certain embodiments, the at least one set consists of a square pyramid truncated with a Vehel angle in each of two opposing trapezoids.

[0094] In certain embodiments, the lateral trapezoids forming the edges of the shell, or the first and second shells, respectively, do not contain a specific pattern of recesses and / or protrusions formed by missing or additional DNA double helix stretches due to specific interactions with complementary patterns on the lateral trapezoids of another one of the self-assembling DNA-based components.

[0095] In certain embodiments, the DNA-based nanostructure comprises (i) a hemi-octahedron T_octa consisting of a set of four copies of a triangular pyramid (FIG. 4A of WO 2021 / 165528), in which a base pair stacking contact on one of the triangular edges of the triangular pyramid is inactivated by either chain shortening or the addition of an unpaired thymidine (see FIG. 4A of WO 2021 / 165528; FIG. 24A,D of WO 2021 / 165528); (ii) a half T=1 shell (see FIG. 4B of WO 2021 / 165528), which consists in each case of two sets of five copies of two different triangular truncated pyramids, the first set of five copies forming a closed pentamer and the second set of five copies docked to the edge of the pentamer (see FIG. 4B of WO 2021 / 165528; FIG. 24B,E of WO 2021 / 165528), and (iii) a "trap" T=1 shell with a missing pentagonal apex (see FIG. 4C of WO 2021 / 165528), which consists in each case of three sets of five copies of three different triangular truncated pyramids, with the first set of five copies forming a closed pentamer, the second set of five copies docked to the edge of the pentamer, the second set of five copies docked to the edge of the pentamer, and the third set of five copies docked in the gap between the second set of five copies (see FIG. 4C of WO 2021 / 165528; FIG. 24C, F of WO 2021 / 165528); (iv) A T=3 icosahedral half-shell consisting of a total of 30 triangular subunits partitioned as five copies of six different full-sized DNA triangular designs with specific edge docking rules. is a shell selected from

[0096] In certain embodiments, the present invention relates to a DNA-based nanostructure further comprising one or more types of DNA brick constructs, each type of such DNA brick construct characterized by one or more interaction sites for specific interaction by edge-to-edge stacking contact with one or more complementary interaction sites respectively present on the plane of the triangular prism or rectangular prism on the outer surface of the DNA-based nanostructure, the DNA brick constructs covering the free space between three or four edges of the plane, respectively (see Figure 33 of WO 2021 / 165528).

[0097] In certain embodiments, the present invention relates to DNA-based nanostructures further comprising one or more bridges within one of said triangular prisms or cuboids, respectively, and / or between two of said triangular prisms or cuboids, respectively.

[0098] In the context of the present invention, the term "bridge" refers to any permanent or intermittent bridge within one of said triangular prisms or cuboids, respectively, and / or between two of said triangular prisms or cuboids, respectively. Any such linkage can be achieved in advance by linking two of the oligonucleotides used to form the self-assembling DNA-based building blocks before assembly, or by chemically or photochemically adding bonds between different parts of the three-dimensional nanostructure, for example. Permanent links can be made, for example, by photochemically crosslinking T residues appropriately placed in the structure under the formation of covalent cyclobutane pyrimidine dimer (CPD) bonds (41), and intermittent links can be made, for example, by photochemically crosslinking the blunt ends of two double helix subunits between a 3-cyanovinylcarbazole (cnvK) moiety placed at the first blunt end and a thymine residue (T) placed at the other blunt end (40).

[0099] In a second aspect, the present invention relates to a composition comprising a DNA-based nanostructure according to the invention encapsulating one or more viruses or viral particles.

[0100] In certain embodiments, the composition is formed by a process of removing the virus or viral particles from a medium containing the virus or viral particles, while in certain other embodiments, the composition is formed by a process of incorporating the one or more viruses or viral particles as cargo into the DNA-based nanostructure.

[0101] 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 said DNA-based nanostructure with a medium containing or suspected of containing said viruses or viral particles.

[0102] In a particular embodiment, (i) a DNA-based half-shell nanostructure based on the T_octa self-assembling DNA-based building block is formed having a size of 50×50×50 nm 3 (ii) DNA-based half-shell nanostructures based on T1_pentamer_triangle self-assembling DNA-based building blocks were selected for viruses with sizes ranging from 15×15×15 to 100×100×100 nm 3 (iii) DNA-based half-shell nanostructures based on a combination of T1_pentamer_triangle and T1_ring_triangle self-assembling DNA-based building blocks were fabricated with sizes ranging from 15×15×15 to 100×100×100 nm 3 and / or (iv) DNA-based half-shell nanostructures based on T3_6_ triangular self-assembling DNA-based building blocks are selected for viruses with a size of 50 × 50 × 50 nm 3 It is selected against viruses of size ≥ 100.

[0103] In certain embodiments, the method is for removing the one or more viruses or viral particles from the medium, hi certain embodiments, the method is for encapsulating the one or more viruses or viral particles for transporting the viruses or viral particles.

[0104] 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 said DNA-based nanostructure with a medium containing or suspected of containing said viruses or viral particles.

[0105] In an alternative aspect, the present disclosure provides a method for encapsulating one or more viruses or viral particles, comprising the step of adding self-assembling DNA-based building blocks to a medium containing or suspected of containing the viruses or viral particles, resulting in the in situ formation of DNA-based nanostructures according to the present invention that encapsulate one or more of the viruses or viral particles.

[0106] In yet another aspect, the present disclosure provides a method for encapsulating a cargo other than a virus or viral particle, such as a complex polymer, 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.

[0107] In an alternative aspect, the present disclosure provides a method for encapsulating a cargo other than a virus or viral particle, e.g., a complex polymer, comprising the step of adding self-assembling DNA-based components to a medium containing or suspected of containing said cargo other than a virus or viral particle, e.g., a complex polymer, resulting in the in situ formation of a DNA-based nanostructure according to the present invention encapsulating said cargo other than a virus or viral particle, e.g., a complex polymer. [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 9-1

Table 9-2

Table 9-3

Table 9-4

Table 9-5

Table 10-1

Table 10-2

Table 10-3

Table 10-4

Table 10-5

Table 10-5

[0108] It is understood that certain features of the invention that are described in the context of separate embodiments for clarity may also be combined and provided in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments relating to the invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and expressly disclosed. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0109] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0110] To the extent possible under the respective patent laws, all patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference.

[0111] The following examples are illustrative of the above invention and are not intended to limit the scope of the invention in any way. Other test models known to those skilled in the art can also determine the beneficial effects of the claimed invention. EXAMPLES

[0112] Introduction Effective broadband antiviral platforms that can act on existing and yet to emerge viruses are not available, creating a need to explore therapeutic strategies beyond the trodden paths. Here, we report virus-encapsulated DNA origami shells that achieve broadband virus capture properties by exploiting the broad background affinity of viruses for heparan sulfate proteoglycans (HSPGs). Calibrated densities of heparan sulfate (HS) derivatives created inside the DNA origami shells allowed us to successfully encapsulate adeno-, adeno-associated, chikungunya, dengue, human papillomavirus, norovirus, poliovirus, rubella, and SARS-CoV-2 viruses or virus-like particles in one and the same HS-functionalized shell system without the need for virus-type specific binders. Our HS-functionalized shells amplify the individually weak reversible interactions of HSPGs to the viral surface by a strong avidity effect that emerges when the curved conformal HS-coated shell envelops the viral particle. Depending on the relative dimensions of the shell to the virus particle, multiple virus particles can be trapped per shell, and multiple shells can also coordinate and surround clusters containing dozens of virus particles. Because steric occlusion in the virus encapsulation shell can prevent the virus from interacting with the host cell, heparan sulfate-coated virus encapsulation shells open an attractive route for establishing broad-spectrum antiviral therapeutic strategies.

[0113] Example 1: Shell design and synthesis principles Here, we address the challenge of creating broad-spectrum antivirals by exploiting the conserved background binding of HSPGs to viruses to irreversibly entrap viruses in a HS-functionalized neutralizing shell (Figure 1A, right panel).

[0114] To capture viruses of different sizes, we created three DNA origami shell variants and functionalized their interiors with the same HS derivative. We used previously described octahedral and T=1 icosahedral half-shell designs (O and T1, respectively; Sigl et al., loc.cit.), which feature cavities of 40 nm and 85 nm width, respectively (Figure 1D). We also developed a new T=3 icosahedral half-shell design, called T3, for the encapsulation of larger virus particles that do not fit into the O and T1 shells (Figure 1E). The T3 design is a finite-sized higher-order assembly of a total of 30 triangular subunits partitioned as five copies of six different full-sized DNA origami triangular designs with specific edge docking rules (Figure 6). The resulting shell has a cavity diameter of approximately 150 nm. Negative stain transmission electron microscopy (TEM) images verify the successful assembly of the T3 shell (Figure 1F and Figure 7).

[0115] Staple strands for origami folding reactions were purchased from Integrated DNA Technologies (IDT) and used with standard desalting purification unless otherwise stated. DBCO-modified handle strands were purchased in HPLC grade from Biomers. 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: customized). VLPs were purchased from The Native Antigen Company, Creative Biostructure, and Creative Biolabs (catalog references can be found in Table 12). [Table 11]

[0116] Folding of DNA origami triangular subunits 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 strand in a standardized "folding buffer" (FoBx) containing x = 20 mM MgCl2, 5 mM Tris base, 1 mM EDTA and 5 mM NaCl at pH 8.00. Scaffold M13 was generated as previously described based on M13 8064 as the scaffold sequence (SEQ ID NO: 1; Engelhardt, FAS et al., ACS Nano 13 (2019) 5015-5027). All folding reactions were subjected to optimized thermal annealing ramps (Table 13) in a Tetrad (Bio-Rad) thermocycling instrument. It should be noted at this point that any variant of M13 8064 or indeed any other single-stranded DNA of sufficient length could have been used as the scaffold sequence with a correspondingly designed set of staple strands. 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 12]

[0117] Purification of triangular subunits and self-assembly of shells All origami structures were purified using agarose gel extraction (1.5% agarose containing 0.5x TBE and 5.5 mM MgCl2) and centrifuged for 30 min at maximum speed to pellet residual agarose. If the origami required a concentration step, ultrafiltration (500 µl Amicon Ultra with a molecular weight cutoff of 100 kDa) was performed prior to shell assembly. For shell assembly, purified triangularis were mixed in a 1:1 ratio. Typical triangularis subunit concentrations ranged from 5 to 400 nM, while assembly times were shell type dependent. 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. [Table 13]

[0118] Assembled shells were UV crosslinked for 1 h at 310 nm using an Asahi Spectra Xenon light source 300W MAX-303 (Gerling, T. et al., Sci. Adv. 4 (2018) eaau1157). Buffer exchange into 1× PBS containing 10 mM MgCl2 was performed prior to VLP encapsulation experiments using ultrafiltration (Amicon Ultra 500 μl with a molecular weight cutoff of 100 kDa) or dialysis (D-Tube™ Dialyzer Mini, MWCO 12-14 kDa, 2× 500 ml exchanges over 8 h, room temperature).

[0119] Heparan sulfate attachment to DNA We used strain-promoted azide-alkyne 1,3-dipolar cycloaddition reaction (SPAAC) to covalently attach heparan sulfate derivatives to DNA oligonucleotides (Figure 1B, Figure 5) that can hybridize to specific acceptor sites in the interior of the DNA origami shell, i.e., single-stranded DNA extensions called "handles". For coupling, we used azide-modified HS derivatives containing either 8 or 18 saccharide monomers (1a and 1c, respectively), including monomers such as N-acetyl-glucosamine and glucuronic acid that characterize HS polymers. As controls, we used 8-mer and 18-mer polysaccharides lacking sulfate and sulfonate groups (1b and 1d, respectively). When the DNA oligonucleotides to be clicked onto the different HS polymers were modified with a dibenzocyclooctyne (DBCO) moiety (2), the SPAAC reaction occurred rapidly upon mixing of both components. We analyzed the reaction products (3a-d) by polyacrylamide gel electrophoresis (PAGE) and revealed different electrophoretic mobilities for the different product versions that were consistent with expectations (Figure 1C). Higher molecular weight reaction products had slower mobility, and sulfate-containing products migrated faster in the gel compared to products lacking sulfate groups, which we attribute to additional negative charges.

[0120] Excess azide-modified heparan sulfate derivatives (1a–d) were mixed with DBCO-modified DNA in a 4:1 ratio to form the respective products (3a–d). MgCl2 was added to a 0.5 M concentration and the mixture was left at 37 °C overnight to achieve >90% conversion. The products were run in a preparative 10% PAGE gel at 35 W for 2 h. The product bands were then excised and crushed. 1× ​​TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8.00) was added to dissolve and recover the modified oligonucleotides, and EtOH precipitation was used for concentration and buffer exchange. The pure products were redissolved and kept in double distilled H2O at either 4 °C or -20 °C.

[0121] Attachment of heparan sulfate-modified DNA constructs to DNA origami shells HS-modified DNA oligonucleotides were then hybridized to the sequence-complementary single-stranded DNA handles protruding from the inner surface of the target DNA origami shell.

[0122] Examination of different heparan sulfate-bound DNA origami shells In initial experiments with adeno-associated virus serotype 2 (AAV2), three different DNA handle designs, namely proximal (H1), distal (H2), and branched (H3), were investigated to determine the type and density of HS modifications required to efficiently capture virus (Figure 1G). These initial experiments with O half-shells showed that H1 was the least efficient, and both the H1 and H2 designs were not as efficient at virus capture as the H3 branched handle design. Samples were analyzed by negative staining 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 to the 18-mer HS derivative (3c), approximately 96% of the shell was occupied by AAV2, improving from the approximately 30 and 84% of occupied shell achieved with H1 and H2, respectively (Figure 8). Thus, we used the branched handle design H3 and the following HS 18-mer variants (3c) unless otherwise specified: We confirmed that the interaction with AAV2 was due to sulfate and sulfonate groups present in the HS structure, since the 3d HS derivative used as a negative control showed no binding (Figure 9). Importantly, all AAV2 particles were captured with an O-shell excess (Figure 10).

[0123] Example 2: Capture of different viruses by DNA origami shells With the HS handle design thus established, we tested the HS-modified DNA origami shells for their ability to capture a variety of exemplary viruses and virus-like particles (VLPs) (Zeltins, A., Mol. Biotechnol. 53 (2013) 92-107). Our targeted virus library sampled enveloped and non-enveloped particles, particles from different virus families, and particles with dimensions ranging from 25 to 90 nm (Table 11, see also Figure 11 for TEM images).

[0124] Maturation of dengue VLPs Maturation of dengue VLPs was adapted 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 VLP samples (10 μl, 0.39 mg / ml, The Native Antigen Company, Cat. No. DENV1-VLP) were added to MES buffer (10 μl, 50 mM, pH 6.00) and mixed gently. Then, CaCl 2(水溶液) (0.75 μl, 0.1 M) and furin (3.9 μl, 2000 U / ml, New England Biolabs, Cat. No. P8077) were added and mixed, and the samples were incubated for 16 hours at 30° C. 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™ Dialyzer Mini, MWCO 12-14 kDa, 2×50 ml exchanges over 24 hours, 4° C.). Mature dengue VLP samples were used immediately and stored at 4° C.

[0125] Virus and VLP encapsulation We used HS-modified O shells to isolate AAV2, poliovirus, mature dengue virus, and norovirus (Figure 2a), HS-modified T1 shells to capture human papillomavirus 16 (HPV16), SARS-CoV-2, chikungunya, and rubella particles (Figure 2b), and HS-modified T3 shells to encapsulate adenovirus 5 (Figure 2c and Figure S12 for TEM tomography).

[0126] Pre-assembled and UV-welded shells in 1x PBS containing 10 mM MgCl2 were mixed with VLP samples in appropriate ratios to achieve either shell or VLP excess. The MgCl2 concentration was adjusted to 10 mM and samples were incubated for 2 h at room temperature. The usual amount of sample for TEM analysis ranges from 5-10 µl of total solution at a triangular origami concentration of approximately 10 nM. Immediately after the 2 h incubation, negative stained TEM grids were prepared.

[0127] Negative staining TEM Samples were incubated on glow-discharged (45 s, 35 mA) formvar carbon-coated Cu400 TEM grids (Electron Microscopy Sciences) for 90–120 s depending on the origami and MgCl2 concentrations. The grids were then stained for 30 s with 2% aqueous uranyl formate containing 25 mM NaOH. Imaging was performed at magnifications of 10,000x–42,000x in Serial EM on an FEI Tecnai T12 microscope operated at 120 kV with a Tietz TEMCAM-F416 camera. TEM micrographs were high-pass filtered to remove long-range staining gradients and contrast was auto-leveled using Adobe Photoshop CS5. Automated grid cell montages were acquired to obtain TEM statistics in an unbiased manner. For detailed information on selected particles, negative stain EM tomography was used as a visualization technique. Tilt series were performed from -50° to +50°, and micrographs were acquired in 2° increments.

[0128] Tilt series were processed with Etomo (IMOD) to obtain tomograms (Kremer, J. et al., J. Struct. Biol. 116 (1996) 71-76). Micrographs were registered to each other by calculating the cross-correlation of successive tilt series images. Tomograms were then generated using filtered backprojection. A Gaussian filter was used with a cutoff between 0.25 and 0.5 and a falloff of 0.035. [Table 14]

[0129] Interestingly, in many cases, the multivalent interactions between the HS coating inside the shell and the virus particle appeared strong enough to support substantial elastic deformation of the surrounding shell. For example, the T3 shell material deformed from a spherical to an elliptical shape around the adenovirus particle, driven by maximizing the number of molecular interactions between the shell inner surface and the HS moieties on the virus surface, presumably at the expense of the shell's elastic deformation. By design, the O shell occasionally deformed to accommodate up to four AAV2 particles within its cavity (Fig. 3a), even though there would only be space for one AAV2 particle if the O shell were completely rigid. The T1 shell also flexed to fit up to three HPV16 copies (Fig. 3b). Depending on the relative stoichiometry between the shell and the virus particle, we also observed sandwich-like structures in which two shells coordinate one virus particle (e.g., with HPV16 and O shells, Fig. 3c). If the shell diameter substantially exceeds the dimensions of the target virus, multiple target particles can be sequestered. For example, we observed up to six AAV2 per T1 shell (Fig. 3d) and up to three chikungunya in a T3 shell (Fig. 3e and Fig. S13 for TEM tomography). Furthermore, multiple copies of HS-modified shells could also cooperatively encapsulate dozens of AAV2 particles in a cluster surrounded and protected by the DNA origami shell material (Fig. 3f). These results support the idea that the shell is flexible to adapt and capture even more pleomorphic viral particles.

[0130] In negative staining TEM images, the Chikungunya VLP particles appeared to completely fill the T1 cavity. Perhaps due to the high degree of shape complementarity obtained, we were able to efficiently capture Chikungunya particles in the T1 shell with high yields using any of the different handle designs described in Figure 1G (H1, 3a, 90% full shell). In fact, we were able to capture Chikungunya even with the 3b negative control, a shell with a coating lacking sulfate and sulfonate groups, albeit with a lower yield (H1, 3b, 54% full shell, see also Figure 14). We interpret this phenomenon as a manifestation of molecular recognition at the mesoscale. This effect is likely due to the cooperative amplification of the weak electrostatic interactions between the negatively charged DNA shell and the Chikungunya particle as they interact over a large surface area. It should be noted that this observation also suggests another route for modification-free virus capture that allows for precise tailoring of the shell to the dimensions of the target virus.

[0131] Dengue virus, as well as several other viruses, exhibit two distinct "mature" and "immature" conformations. To become infectious, viral surface proteins must undergo certain conformational changes that allow them to move between vector and host and / or between infected 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 Martin, C., Virus Maturation. In: Physical Virology: Virus Structure and Mechanics(ed. Greber, UF)129-158(Springer International Publishing,2019), doi:10.1007 / 978-3-030-14741-9_7). Although the use of VLPs is very convenient for safety reasons, we acknowledge some limitations. For example, initially, our dengue VLP samples contained a high proportion of immature particles that did not bind to our HS-functionalized shell. To overcome this, we induced enzymatic maturation of dengue VLPs and observed binding of mature particles, as would occur in vivo (Fig. 2a, dengue, and Fig. 15).

[0132] We also performed cryo-electron microscopy (cryo-EM) measurements of HPV16 and Chikungunya VLPs entrapped within the O and T1 shells, respectively (Figure 4).

[0133] Cryo-EM DNA origami shells were prepared and functionalized, and virus was captured as described above. Samples (O+HPV: 70 nM triangular; T1+Chikungunya: 200 nM triangular) were incubated for 60 s on glow-discharged lacey carbon 400 mesh copper grids with ultrathin carbon films. Grids were subsequently plunge-frozen in liquid ethane using an FEI Vitrobot Mark V (blot time: 2.5 s, blot force: -1, drain time: 0 s, 22 °C, 100% humidity, 3 μl sample). Cryo-EM imaging was performed using a spherical aberration (Cs)-corrected Titan Krios G2 electron microscope (Thermo Fisher) operated at 300 kV and equipped with a Falcon III 4k direct electron detector (Thermo Fisher). Automated image acquisition was performed using EPU 2.6 (dose: 42–45 e - / Å 2 , exposure time: 3-5 s, 12 fractions, pixel size: 0.23 nm (O+HPV) and 0.29 nm (T1+Chikungunya), defocus: -1.5 to -2 μm). Micrographs were processed in RELION-3 (Zivanov, J. et al., eLife 7 (2018) e42166) using MotionCor2 (Zheng, S. et al., Nat. Methods 14 (2017) 331-332) and CTFFIND4.1 (Rohou, A. & Grigorieff, N., J. Struct. Biol. 192 (2015) 216-221). Particles were automatically collected with cryYOLO 1.7.6 (Wagner, T. et al., Commun. Biol. 2 (2019) 1-13). Extracted particle images were classified and selected by visual inspection with multiple rounds of 2D and 3D classification. Initial models were generated in silico in RELION-3. 3D reconstruction and multibody refinement yielded electron density maps with a resolution of 26 Å for the O shell capturing HPV (EMD-13884, 1×O+HPV: 7834 particles, 2×O+HPV: 4634 particles) and 36 Å for the T1 shell capturing Chikungunya (EMD-13883, 1259 particles, C5 symmetry).

[0134] Two-dimensional (2D) class average images and 3D cryo-EM reconstructions confirmed that the VLPs were successfully entrapped within the cavities of their respective shells (Fig. 4b, e and Fig. 16-17). One O shell is not large enough to encapsulate the entire HPV16 particle, but two O copies are able to coordinate and completely cover the entire VLP (Fig. 4c). 2D class averages of free HPV16 showed particle size variation within the VLP samples (Fig. 18). Consistently, we also found that the gap distance between the O shells (indicated by the white arrows in Fig. 4b) changed depending on whether smaller or larger HPV16 particles were entrapped. The cryo-EM maps we determined for the complex consisting of chikungunya VLPs within the HS-modified T1 shell reveal a near-perfect fit between the two particles (Fig. 4e, f). The cryo-EM maps provide a compelling illustration of the relative dimensions of the artificial DNA origami shells to the viral target, and the degree of surface occlusion that can be achieved by sequestering the virus within the shell.

[0135] Example 3: Stability of DNA origami shells encapsulating virus / VLPs Finally, to test the stability of virus capture by the shell, we exemplarily subjected a sample consisting of AAV2 encapsulated in an O-shell to a dilution series. The percentage of occupied shells remained the same before and after 100-fold dilution and 14 days of incubation in the diluted samples compared to the undiluted samples (Figure 19), suggesting that the spontaneous dissociation rate of the complex formed between the AAV2 particles and the surrounding HS-modified shell is at least on the scale of weeks under the conditions tested. The high stability is avidity-driven and can be understood by considering that spontaneous dissociation of AAV2 from the surrounding shell requires the simultaneous destruction of several dozen bonds formed between the HS chains on the enveloping shell and the viral surface. The probability of such an event occurring decreases exponentially with the number of HS bonds formed.

[0136] Example 4: Encapsulation efficiency of virus / VLPs by DNA origami shells 1-Handle design and development for efficient virus capture To optimize and calibrate the density required for our heparan sulfate derivatives, we exemplarily investigated the capture efficiency of AAV2 with an O half-shell using three different handle variants (Figure 1G and Figure 8a). The proximal handle (H1) was the shortest design tested and consisted of a DNA stretch of 26 nucleotides, placing the heparan sulfate modification in a proximal configuration. The distal handle design (H2) contained a single-stranded stretch of 20 thymidines (poly-T stretch), allowing the heparan sulfate groups to reach further from the origami surface and increase the likelihood 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.

[0137] The three handle designs were tested in parallel, with O half shells and excess AAV2 particles. Samples were analyzed by 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 versus empty shells for all three handle variants hybridized to heparan sulfate 3c. These experiments revealed that H1 was not as efficient at virus capture as the longer and denser H2 and H3 handles. With H1, only 20% of the O shell was occupied by AAV2, whereas with H2 and H3, capture increased to 84% and 96%, respectively (Figure 8b). Unless otherwise stated, the branched handle design (H3) hybridized to heparan sulfate 18-mer variant 3c was used below.

[0138] 2- Multiple virus capture and entrapment of different virus types in the very same shell unit To test whether our system can be used as a true broad-spectrum virus capture platform, heparan sulfate modified T1 half shells were subjected to capture of a cocktail of viruses consisting of AAV2, HPV16 and Chikungunya particles. Negative stain TEM characterization of such samples revealed capture of all virus types present in the cocktail (Figure 21). Field micrographs in Figures 21A and B illustrate the good performance of the system. Some shells were found to encapsulate individual viruses such as one Chikungunya particle (Figure 21C), one HPV16 (Figure 21D) and one AAV2 (Figure 21E), but multiple particles were also simultaneously captured in the exact same shell unit as seen with several AAV2 (Figure 21F top), HPV16-Chik (Figure 21F bottom), AAV2-Chik (Figure 21G) and AAV2-HPV16 (Figure 21H).

[0139] 3. Collaborative shell capture of virus clusters When the shell diameter was substantially larger than the dimensions of the target virus, we observed that multiple virus particles could be sequestered. Interestingly, multiple copies of the HS-modified shell could partition and cover the surface of the AAV2 cluster (Figure 22).

[0140] summary: In conclusion, here we present a virus capture system that targets viral features conserved across many families through the use of HS derivatives. Overall, we achieved encapsulation of nine different viruses and VLP test samples, each representing a different virus family and exhibiting different sizes and surface complexities. Our modular shell system creates a locally curved environment within the cavity that allows for highly multivalent binding and can be optimized according to size and ligand density / type to realize a broad-spectrum antiviral platform that binds irreversibly. Our shells can bend and adapt to the shape of the captured virus particles to some extent, suggesting that the shell system can also accommodate pleomorphic virus particles.

[0141] We envision that our HS-modified DNA origami shells can function as cell surface decoys, capturing the virus and preventing its interaction with the cell surface, thus reducing the effective viral load in acute infections. Testing the therapeutic potential of this system to reduce viral load in vivo remains an important future challenge. Beyond virus neutralization, our system may also function as a sink to capture associated viral proteins (Figure 20) and other by-products 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 shell library has the potential to be a suitable therapeutic platform to combat viral infections.

Claims

1. DNA-based nanostructures, The DNA-based nanostructure is a shell having a cavity surrounded by the DNA-based nanostructure, The DNA-based nanostructure is formed from self-assembling DNA-based components, Each of the self-assembling DNA-based components is formed by a single-stranded DNA template strand and a set of oligonucleotides complementary to the single-stranded DNA template. Each of the oligonucleotides is complementary to either one continuous DNA sequence stretch or at least two discontinuous DNA sequence stretches on the single-stranded DNA template. Each of the self-assembling DNA-based components is a triangular prism and / or a rectangular prism, in particular a triangular prism, Each subset of one or more oligonucleotides in one or more of the self-assembling DNA-based components is linked to a construct containing at least one sulfonated or sulfated polysaccharide group toward the interior of the cavity, particularly a construct containing one or two sulfonated or sulfated polysaccharide groups, Each construct comprises (i) a handle having at least one binding site for the sulfonated or sulfated polysaccharide group, and (ii) the sulfonated or sulfated polysaccharide group(s) attached to the handle, wherein the handle has a length corresponding to the length of a single-stranded oligonucleotide containing at least 30 nucleotides.

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

3. The DNA-based nanostructure according to claim 1, wherein each of the sulfonated or sulfated polysaccharide groups is independently selected from the list of 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, particularly heparan sulfate.

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

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

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

8. The DNA-based nanostructure according to claim 1, which is a closed three-dimensional geometric shape, particularly a sphere, a spherical cylinder, and a polyhedron, particularly a tetrahedron, octahedron, or icosahedron, formed in situ from the self-assembling DNA-based components in the presence of the encapsulated virus or viral particles.

9. The DNA-based nanostructure according to claim 1, wherein the shell has an opening for accessing the cavity.

10. The DNA-based nanostructure according to claim 1, comprising a combination of first and second subshells, each having an opening for accessing first and second internal cavities, wherein the first and second internal cavities together form the cavity, and in particular the first and second subshells are connected by at least one linker.

11. A DNA-based nanostructure according to claim 1, 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 of self-assembling DNA-based components, and each of the self-assembling DNA-based components is a triangular prism and / or a rectangular parallelepiped, particularly a triangular prism.

13. Each of the aforementioned triangular prisms and / or rectangular parallelepipeds is formed by m triangular or rectangular planes, where m is an integer independently selected from 4, 5, 6, 7, and 8, particularly independently selected from 5, 6, and 7, and more specifically, the integer is 6. Each of the three or four edges of the m planes is formed by n parallel stretches of the DNA double helix, where n is an integer independently selected from 1, 2, 3, 4, 5, and 6, particularly independently selected from 2, 3, 4, and 5, and more specifically independently selected from 3 and 4. Each plane is connected to a plane above and / or a plane beyond the plane by (i) stacking the interactions between the DNA double helices that form the plane, and (ii) DNA stretching in the oligonucleotides that form the DNA-based components that bridge the single-stranded DNA template and / or at least two of the planes, The DNA-based nanostructure according to claim 12, wherein at least two of the three or four lateral trapezoids each include a specific pattern of recesses and / or protrusions formed by missing or additional DNA double helix stretches for specific interaction with a complementary pattern on another lateral trapezoid of the self-assembling DNA-based component.

14. The DNA-based nanostructure (a) A hemioctahedral DNA-based nanostructure based on T_octa self-assembling DNA-based components, comprising a set of four copies of a frustum of a triangular pyramid, wherein the base pair stacking junction at one of the triangular edges of the frustum of the triangular pyramid is inactivated by either chain shortening or the addition of unpaired thymidine, (b) T1_Pentamer_Triangular self-assembling DNA-based components, each consisting of two sets of five copies of two different truncated triangular pyramids, the five copies of the first set forming a closed pentamer, and the five copies of the second set docked onto the edge of the pentamer, and (c) Half shells of "trap" T=1 with missing pentagonal vertices, based on a combination of self-assembling DNA-based components of T1_pentamer_triangle and T1_ring_triangle, each consisting of three sets of five copies of three different frustums, the five copies of the first set forming a closed pentamer, the five copies of the second set docking to the edge of the pentamer, the five copies of the second set docking to the edge of the pentamer, and the five copies of the third set docking to the gaps between the five copies of the second set, (d) A DNA-based nanostructure according to claim 1, a T=3 icosahedral half-shell based on a T3_6 triangle-based self-assembling DNA-based component, the half-shell being selected from a T=3 icosahedral half-shell comprising a total of 30 triangular subunits partitioned as five copies of six different full-size DNA triangle designs having specific edge docking rules.

15. (a) One or more types of DNA brick constructs, each type of such DNA brick construct characterized by one or more interaction sites for specific interactions by inter-edge stacking contact with one or more complementary interaction sites located on a frustum-of-triangular or frustum-of-square plane on the outer surface of the DNA-based nanostructure, wherein each DNA brick construct covers the free space between three or four edges of the plane. (b) One or more bridges in one of the triangular prisms and / or rectangular parallelepipeds, and / or between two of the triangular prisms and / or rectangular parallelepipeds, and / or (c) The DNA-based nanostructure according to any one of claims 12 to 14, further comprising at least one portion that specifically interacts with the virus or viral particles.

16. A composition comprising a DNA-based nanostructure according to any one of claims 1 to 14, which encapsulates one or more viruses or viral particles.

17. A method for encapsulating one or more viruses or viral particles, comprising the steps of: providing a DNA-based nanostructure according to any one of claims 1 to 7 and 9 to 14; and contacting the DNA-based nanostructure with a culture medium containing or suspected to contain the virus or viral particles.

18. (i) DNA-based half-shell nanostructures based on T_octa self-assembling DNA-based components, 50 × 50 × 50 nm ３ Selected for viruses up to (ii) T1 pentamer triangular self-assembling DNA-based components, DNA-based half-shell nanostructures are available in sizes from 15 × 15 × 15 to 100 × 100 × 100 nm. ３ Selected for viruses of the following size, DNA-based half-shell nanostructures based on a combination of self-assembling DNA-based components of (iii) T1_pentamer_triangle and T1_ring_triangle are used in sizes from 15×15×15 to 100×100×100 nm. ３ Selected for viruses of the size (iv) T3_6_, a DNA-based half-shell nanostructure based on triangular self-assembling DNA-based components, is 50 × 50 × 50 nm. ３ The method according to claim 17, which is selected for viruses of the above size.

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